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The Effect of Putative Vesicular Stomatitis Virus Methyltransferase Mutants on Transcription and Replication

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PAGE 1

THE EFFECT OF PUTATIVE VESICULAR STOMATITIS VIRUS METHYLTRANSFERASE MUTANTS ON TRANSCRIPTION AND REPLICATION By DALLAS L. TOWER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Dallas L. Tower

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iii ACKNOWLEDGMENTS First and foremost, I would like to thank my parents for supporting me in all my endeavors and instilling in me the strong desi re to accomplish all of my goals. I owe a tremendous debt of gratitude to my pare nts for their encourag ement and assistance throughout my academic career. I thank my lab members, Sherin Smallwood, Valery Grdzelishvili, and Dorothy Smith, for thei r assistance, guidance, and friendship throughout my tenure at the University of Fl orida. I would like to thank my committee members, Sue Moyer, Donna Duckworth, and Richard Condit, for their advice on my project. My advisor, Sue Moyer, has been an incredible mentor to me over the past three years. I thank her for her patience and guidan ce. She has instilled in me a deep respect for scientific researchers and I cannot tha nk her enough for the edu cation I have received under her counseling.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................vi ii CHAPTER 1 INTRODUCTION........................................................................................................1 General Background.....................................................................................................1 Transcription.................................................................................................................2 Replication.................................................................................................................... 3 Assembly and Release..................................................................................................3 Proteins....................................................................................................................... ..4 M Protein...............................................................................................................4 G Protein................................................................................................................4 N Protein................................................................................................................4 P Protein................................................................................................................5 L Protein................................................................................................................5 Capping and Methylating Mechanism..........................................................................8 Project Background......................................................................................................8 2 MATERIALS AND METHODS...............................................................................11 Clones......................................................................................................................... 11 Cells and Virus...........................................................................................................15 TNT Coupled Transcription/Tr anslation Reaction System........................................16 In vivo Expression of L Mutant Proteins....................................................................16 CAT (Chloramphenicol Acetyltransferase) Assay.....................................................17 3 RESULTS: HR 1..........................................................................................................21 Clones......................................................................................................................... 21 TNT Coupled Transcription/Translati on Reaction Analysis of L Proteins................22 In vivo Expression of L Mutants................................................................................23 Analysis of the hr 1 L Mutants for Transcription and Replication..............................23

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v 4 RESULTS: HR8 ..........................................................................................................29 Clones......................................................................................................................... 29 TNT Coupled Transcription/Translati on Reaction Analysis of L Proteins................29 In vivo Expression of L Mutants.................................................................................31 Analysis of the hr 8 L Mutants for Transcription and Replication..............................32 5 DISCUSSION.............................................................................................................38 LIST OF REFERENCES...................................................................................................44 BIOGRAPHICAL SKETCH.............................................................................................48

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vi LIST OF TABLES Table page 1-2 Nomenclature of hr 1 mutants.....................................................................................10 1-3 Nomenclature of hr 8 mutants.....................................................................................10 2-1 Primers used to inco rporate the mutations of hr 1 and hr 8..........................................13 2-2 Mutagenic primers and incorpor ated silent rest riction sites........................................14 2-3 Subcloning enzymes, vectors, and inserts...................................................................15 3-1 CAT synthesis of hr 1 mutants in HEp-2 cells at 34C...............................................26 3-2 CAT synthesis of hr 1 mutants in HEp-2 cells at 37C...............................................26 3-3 CAT synthesis of hr 1 mutants in HEp-2 cells at 40C...............................................26 3-4 CAT synthesis of hr 1 mutants in A549 cells at 37C................................................27 3-5 CAT synthesis of hr 1 mutants in BHK cells at 37C.................................................27 3-6 Comparative tite rs of recombinant VSV hr 1 mutants in BHK and HEp-2 cell lines at 34C and 40C..................................................................................................... 28 4-1 CAT synthesis of hr 8 mutants in HEp-2 cells at 34C...............................................34 4-2 CAT synthesis of hr 8 mutants in HEp-2 cells at 37C...............................................34 4-3 CAT synthesis of hr 8 mutants in HEp-2 cells at 40C...............................................35 4-4 CAT synthesis of hr 8 mutants in A549 cells at 37C................................................36 4-5 CAT synthesis of hr 8 mutants in BHK cells at 37C.................................................36 4-6 Comparative tite rs of recombinant VSV hr 8 mutants in BHK and HEp-2 cell lines at 34C and 40C......................................................................................................37

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vii LIST OF FIGURES Figure page 1-1 Schematic of the VSV gene order..............................................................................3 1-2 Conserved regions in Sendai L protein......................................................................7 2-1 The pBS-SK-L plasmid used as th e WT-L template for cloning through mutagenesis..............................................................................................................13 2-2 Representation of the DI-CAT.................................................................................19 2-3 Representation of the transformation of the CAT ORF into a functional mRNA...19 2-4 Representation of antibody bi nding in Roche CAT-ELISA kit...............................20 3-1 TNT of hr 1-0 and other hr mutants..........................................................................22 3-2 In vivo expression of hr 1 L-mutants........................................................................23 4-1 TNT of hr 8-1,2 and hr 8-3........................................................................................30 4-2 TNT of hr 8-1,2 and hr 8-1,2,3 subclones.................................................................31 4-3 TNT of hr 8-4 and hr 8-1,2,3,4..................................................................................31 4-4 In vivo expression of hr 8 L-mutants........................................................................32 5-1 Conserved domains in VSV and the location of the hr 1-1 mutation.. .....................41

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viii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE EFFECT OF PUTATIVE VESICULAR STOMATITIS VIRUS METHYLTRANSFERASE MUTANTS ON TRANSCRIPTION AND REPLICATION By Dallas L. Tower May 2005 Chair: Sue A. Moyer Major Department: Molecular Genetics and Microbiology The vesicular stomatitis virus (VSV) mRNAs are methylated at the guanine-N7 and 2’-O-adenosine positions. The RNA-depende nt RNA polymerase synthesizes the viral mRNAs which are modified at their 3’ and 5’ ends by polyadenylation, capping, and methylation. Two host range mutants, hr 1 and hr 8, isolated by Simpson and Obijeski are defective in methylation. WT mRNA caps are dimethyl ated by 7-methylguanosine and 2’-O-methyltransferase yielding the cap structure 7mGpppAmpAp. The cap on an mRNA from hr 1 has no methylation (GpppApAp) and the cap on an mRNA from hr 8 was thought be 2’-O-mono-methyl ated or not methylated at all. Unmethylated mRNAs, like those produced by hr 1 and hr 8, are deficient in protein synthesis. Further, hr 1 and hr 8 are host restricted as well as temperature se nsitive. The N, P, and L proteins that form the polymerase and the respective genes of the hr 1 and hr 8 mutants were sequenced

PAGE 9

ix and compared to WT N, P and L genes. Hr 1 has two amino acid substitutions differing from WT in the L gene and hr 8 has five amino acid substitutions, also found in the L gene. In this study, the hr 1 and hr 8 mutations were introduced separately, together, and combination into the L gene. The effects on the VSV L mutant and WT L proteins were studied in CAT minigenome assays. The assays were conducted in permissive and nonpermissive cell lines as well as permissi ve and nonpermissive temperatures. The second amino acid change at position 1671 in the hr 1 mutant appears to be responsible for the temperature sensitivity. The data on the hr 8 mutant proved to be inconclusive. Further studies performed in the Moyer lab confirm that amino acid 1671 is responsible for temperature sensitivity as well as th e host restriction. Additionally, amino acid position 1481 is responsible for both host restri ction and temperature sensitivity. Position 1671 may play a critical role within the S-adenosylmethionine binding domain and position 1481 could possibly be part of the catalytic site for 7mG methyltransferase.

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1 CHAPTER 1 INTRODUCTION General Background Vesicular Stomatitis Virus (VSV) is a Vesiculovirus found in the order Mononegavirales. A number of viruses causing huma n diseases are contained in this order, including measles, mumps, rabies, E bola, Marburg, Borna, Hendra, and Nipah. More specifically VSV is found in the family Rhabdoviridae, which al so includes rabies. Over 200 rhabdoviruses have been identified. Vesiculovirus is one of six genera in the Rhabdoviridae family. VSV contains two serotypes, Indiana and New Jersey, both of which infect a broad range of insects and mammals. The virus is present in North and South America and relatives of the virus can be found in Africa a nd Asia. Symptoms of the disease resemble foot and mouth disease with vesicular lesi ons on the tongue, gums, teats and hooves in livestock. Headache, nausea, malaise and ge neral influenza-like symptoms are also possible. The virus is spread through vesicular fluid and saliva; it can also be transmitted by infected flies and mosquitoes. VSV can infect humans causing a mild illness. VSV, like rabies, is a bulle t shaped virus. It is approximately 180 nm long by 75 nm wide. The virus is enveloped with G-prot ein spikes on the surface. It has a singlestranded negative sense RNA ge nome of 11,200 nucleotides. R NA that is negative sense cannot be translated immediately after entry into the cell. Therefore, an antigenomic intermediate must be produced by the R NA-dependent RNA polymerase, which comes packaged in the virus, in or der to make a viable mRNA.

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2 Transcription Before transcription can occur, the virus must attach to a receptor on a host which initiates the infection. Next, the virion is endocytosed by the host cell and the ribonucleoprotein (RNP) core with the attached RNA-dependent RNA polymerase (RdRp) is released into the cytoplasm. The RNP with N-encap sidated, negative strand RNA serves as the template for transcripti on. The RdRp, which consists of two viral subunits: the phosphoprotein (P) and the large pr otein (L), transcribes the template to produce (+) strand mRNAs which are modifi ed at their 3’ and 5’ ends by polyadenylation, capping, and me thylation, respectively. The viral genes are transc ribed sequentially in VSV, producing leader RNA and then the capped and methylated mRNAs with a gene order of 5’-N-P-M-G-L-3’ (Ball and White, 1976) (Figure 1-1). VSV polymerase paus es between each gene and then begins synthesizing the next downstream gene (Iv erson and Rose, 1981). Possibly, this is to generate the poly(A) tail on the mRNA. Additionally, the polymerase is responsible for capping the mRNA (See Capping and Methylating Mechanism). It is thought that conserved sequences preceding and within each gene contain the capping and re initiation signals (Stillm an and Whitt, 1999). Cap methylation, involving the methyl donor S-adenosyl-L-methionine (AdoMet), is essential for translation of viral mRNAs but not mandatory in viral mRNA synthesis (Gingras et al., 1999, Horikami et al., 1984, Horikami and Moyer 1982).

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3 Figure 1-1. Schematic of the VSV gene order. Image taken from Rose and Whitt (2001). Replication Replication of VSV occurs after the synthesis of mRNA s and their translation. Replication requires ongoing protein synthesis. Genomic replication cannot occur until N protein is synthesized. Add itionally, the P and L proteins, which form the polymerase catalyzing transcription, are also responsible for replication. Sufficient encapsidation of leader RNA by the N protein signals the polymerase to ignore mRNA initiation and termination signals and consequently pr oduces full length positive-sense genome RNA (Blumberg et al., 1983). Genome RNA synthesi s is always coupled to its encapsidation by N. The encapsidated positive-stranded ge nome RNA is replicated again to produce the encapsidated genomic negative-stranded RNA. Assembly and Release Encapsidated RNA (RNP) is now ready for assembly and release. The matrix protein (M) condenses the RNP and packag es it along with the RNA-dependent RNA polymerase. The glycoprotein (G), whic h produces the spikes on the surface of the virus, is transported through th e secretory pathways of the cell to the outer cell plasma membrane. In VSV, this is typically on the basolateral surface of cells (Stephens et al., 1986). The M protein brings the assembled RNP and viral polymerase complex to the

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4 cell plasma membrane through an interaction of the G and M proteins. As the virus buds out from the cell membrane, the glycoprotein spikes become the new exterior of the virus. Proteins M Protein The M protein, or matrix protein, is derive d from the third mRNA to be transcribed from the viral genome. M is the smallest prot ein of the five VSV proteins. It condenses the nucleocapsid for packaging, disrupts the cytoskeleton, and aids the budding of the virus from the cell. G Protein The G protein, or glycoprotein, is derived from the fourth mRNA to be transcribed from the viral genome. It is a membrane prot ein responsible for the coat of spikes (~400) on the virion membrane. The G protein enha nces the efficiency of budding 10to 30fold (Rose and Whitt, 2001). G protein attaches the virus to the susceptible cell and is the protein to which neutralizing antibody is directed. N Protein The N protein, or nucleocapsid protein, is derived from the first mRNA to be transcribed from the viral genome. N is a cy toplasmic protein with several functions. N is responsible for packaging the RNA into a compact core that serves as the template for transcription and replication. The P-L pol ymerase complex binds to N on the RNP during transcription and repl ication and N interacts with the M protein during virus assembly. As mentioned previously, N is thoug ht to control the rates of transcription and replication as well as the transition between transcription and repl ication (Blumberg and Kolakofsky, 1981). The N protein probably has two domains: the highly conserved

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5 (~80%) N-terminus and the poorly conserved (~20%) C-terminus (Parks et al., 1992). Deletion experiments by Buchhol z et al. (1993) suggest that the highly conserved Nterminal region is required for RNA binding and nucleocapsid assembly. The P protein of the polymerase complex binds the C-terminus of N in RNP. P Protein The P protein, or phosphoprotein, is deri ved from the second mRNA to be transcribed from the viral genome. The P protein was initia lly thought to be a homotrimer, but more recent studies indicate th at it is actually a tetr amer (Tarbouriech, et al., 2000). P works in combination with th e L protein in forming the RNA-dependent RNA polymerase. It alone is not responsible for enzymatic activity, but probably helps fold the L protein for activity (Kolakofsky et al., 1991). Additionally, P binds to N and preserves the soluble state of N in the cy toplasm until RNA genome synthesis requires N for encapsidation (Robbins and Bussell, 1979). The coiled-coil oligomerization regions of the P protein in the C-terminal end are necessary for the interacti on between P and L (Ryan and Portner, 1990). P protein oligomerization occurs in many viruses, such as respiroviruses, morbilliviruses, Rubulavirus, and RSV (Slack and Easton, 1998). Therefore, oligomer ization is thought to be a general characteristic of the protein. The N-terminus contains a small region th at is essential for RNA encapsidation. The specific function of the region is unknown. However deletion of residues 1 to 78 of the P protein hinders RNA synthesis a nd encapsidation (Curran et al., 1994). L Protein The L protein, named for its large size, is derived from the fifth mRNA to be transcribed from the viral genome. Like P, the L protein is also an oligomer (Smallwood

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6 et al., 2002, Cevik et al., 2003) Containing more than 2100 amino acids in a single polypeptide chain, it is the largest of the five VSV proteins. The L pr otein is the catalytic subunit of the RNA-dependent RNA polymerase. It is a multifunctional protein involved in transcription, replication, mRNA capping, methylation, and polyadenylation (Abraham et al., 1975, Hercyk et al., 1988, Schubert et al., 1980). Due to its multifunctional characteristics, it was believed that the L protein consisted of several functional regions. Fu rther experiments confirmed this hypothesis. When L protein sequences from several ( -) sense RNA viruses were compared, six conserved regions were found and named Domain s I to VI (Poch et al., 1990, Sidhu et al., 1993) (Figure 1-2). Domains II, III and VI c ontain conserved motifs that appear to be essential for RNA synthesis. Domain II contains a charged RNA binding motif (Smallwood et al., 1999). A template recognition/phosphodiester bond-forming motif, mandatory for VSV RNA synthesis, has been mapped to Domain III (Jin and Elliot, 1992, Sleat and Banerjee 1993, Schnell and Con zelman, 1995). In 1993, Canter et al. showed that a purine binding element essen tial for polymerization could be eliminated with one deletion in Domain VI of VSV L. Additional studies by Feller et al., 2000, show ed that when mutations are inserted in Domains IV and/or Domain VI a broad ra nge of RNA synthesis defects occur. For example, Sendai virus L protein becomes h eat sensitive and can no longer transcribe effectively. Some of the mutations resulted in a decrease in le + RNA synthesis and even a lack of RNA synthesis initiation. However, mutations in Domain IV and VI of the L protein in Sendai virus did not re sult in the inability of L to bind P. Mutations in Domain

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7 V also gave multiple defects in RNA synthesis, however the majority of the mutants were defective in replication but not tr anscription (Cortese et al., 2000). The original thought was that each of th e six conserved regions of the L protein corresponded to a separate activity (Poch et al., 1990, Sidhu et al., 1993). However, when Smallwood et al. (2002) and Cevik et al (2003) used site-directed mutagenesis to create mutants in each of the domains, th ey noticed a commonality of RNA synthesis defects between each of the domains. Therefore, similar phenotypes can be attained through multiple mutations across the domains suggesting multiple independent domains. This was supported by the finding that two L prot eins that are defici ent in RNA synthesis alone can complement each ot her and restore RNA synthesis in vitro and this complementation is dependent on cotranslation of the L mutants, suggesting that L is an oligomer in the polymerase complex (Smallwood et al., 2002). Figure 1-2. Conserved regions in Sendai L pr otein. Amino acid positions are indicated above the beginning of the conserved region. Image taken from Feller et al. (2000).

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8 Capping and Methylating Mechanism Abraham et al. (1975) suggest that capping is mediated by the polymerase complex. But, so far no mutations located in the L protein have affected capping. However, defects in viruses with a methyltran sferase deficiency have been mapped to the L protein (Hercyk et al., 1988) Cellular mRNA caps are formed by essentially three enzymatic steps. First, RNA triphosphatase removes the phosphate from the 5’ end of the nascent RNA. Then, guanylyltransferas e adds a GMP in a 5’ppp5’ linkage to the nascent RNA. Lastly, methyl groups are ad ded at G and the penultimate base forming the 7mG and 2’O base. However, 5’ ca pping in non-segmented negative strand (NNS) RNA viruses is different. The and phosphates in the GpppA phosphate bridge are derived from a GDP donor (Abraham et al., 1975) which is added to GMP terminated RNA. Then, the 5’ terminus is methyl ated by guanine-N7-methyltransferase and nucleoside-2’-O-methyltransferase activities of L (Moyer and Banerjee, 1975) to yield m7GpppAmpNpNpNp. Project Background In the 1970’s, Simpson and Obijeski were chemically mutagenizing VSV to determine functional and structural properties of the viral genus (Simpson and Obijeski, 1974). Since VSV normally has such a broad host range, they attempted to isolate mutants that exhibited host restriction ( hr ). VSV hr mutants share the same structural proteins as WT VSV. Many of the mutants we re host restricted in cells of human origin, like HEp-2, or HeLa cells, but not in chicken embryo fibr oblasts or hamster cells. Hr 1, for example, had deficient protein synthesi s in nonpermissive cells. Some of the hr mutants appeared to be temperature sensitive as well.

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9 Normal viral mRNAs that are synthesized in vitro or in vivo are polyadenylated at the 3’ end and guanylated and methylated at th e 5’ end. However, further studies on the hr mutants have shown that hr 1 is totally defective and hr 8 is partially defective in vitro in methylating the mRNA (Horikami and M oyer, 1982). A WT mRNA cap structure is presumed to be methylated by two ac tivities, 7-methylguanosine and 2’-Omethyltransferase in L, yielding a dimet hylated mRNA: 7mGpppAmpAp. The cap on an mRNA from hr 1 has no methylation, GpppApAp, and the hr 8 mRNA cap can be 2’Omono-methylated or not methylated at all. 7mG methylated mRNAs are required for protein synthesis in mammalian cells, so unmet hylated mRNAs give the deficient protein synthesis described by Obijeski and Simps on (Simpson and Obijeski, 1974, Horikami and Moyer, 1982). The L protein was shown to possess the met hyltransferase activi ties (Hercyk et al., 1988) and sequencing of the VSV L, N, and P genes has shown that these hr mutations are in fact in the L protein. Hr 1 has two amino acid changes from WT L and hr 8 has five changes (Table 1-1). The goal of my work is to determine which one amino acid change, or combination of changes, is actually res ponsible for the host restriction and temperature sensitivity of the viruses by measuring the e ffect each mutation in L has on transcription and replication when compared to WT L. Ea ch amino acid change has been constructed separately in the WT L gene, and named as shown in Tables 1-2 and 1-3. Additionally, several combinations of changes have also be en made in the WT L gene. The original L mutants of hr 1 and hr 8 have also been reproduced by the combination of all the mutations.

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10 Table 1-1. Amino acid changes of hr 1 and hr 8 from Simpson WT. Amino Acid Simpson WT hr 1 hr 8 148 THR ALA 505 ASN ASP 1097 THR ILE 1356 ALA ASP 1375 TYR SER 1481 GLY ARG 1671 ASP VALTable 1-2. Nomenclature of hr 1 mutants. NomenclatureAmino Acid Simpson WT hr 1* hr1-0 505 ASN ASP hr1-1 1671 ASP VAL *The original hr 1 mutant contains both amino acid changes from Simpson WT. Table 1-3. Nomenclature of hr 8 mutants. NomenclatureAmino Acid Simpson WT hr 8* hr8-0 148 THR ALA hr8-1 1097 THR ILE hr8-2 1356 ALA ASP hr8-3 1375 TYR SER hr8-4 1481 GLY ARG *The original hr 8 mutant contains all five mutati ons. Each individual mutation has a name that corresponds with its amino acid change from Simpson WT.

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11 CHAPTER 2 MATERIALS AND METHODS Clones In general, all the clone s were produced by making a PCR product where the WT VSV L gene or sequential hr mutant L genes were used as the template and two complementary mutagenic primers (Table 2-1) with a silent restriction site created the desired mutation. The pBS-SK-L plasmid (F igure 2-1) was used as the WT VSV L template. The pBS-SK-L plasmid, as well as the pBS-N and pBS-P plasmids, was kindly provided by J. Rose. The Stratagene QuikCh angeXL Site-directed Mutagenesis Kit was used to direct the PCR. The PCR product wa s digested with DpnI and then transformed into the ultracompetent cells provided by the kit. The cells were plated on LB-Amp+ plates and colonies were then screened by PCR with primers bracketing the mutation site and that PCR product was digested with an enzyme that corresponde d to the introduced silent restriction site. The si lent restriction site was desi gned so as not to change the amino acid sequence, but allow a cut site near the desired mutation to suggest that the mutation was also present (Table 2-2). Coloni es that contained the cut site were grown up, and the DNA was isolated by a Qiagen Mi di-prep or Maxi-prep. A TNT coupled transcription/transla tion (see below) was performed on the DNA to make sure that it produced a full length protein and the DNA was sequenced by UF’s DNA Sequencing Core to determine that it was actually correct. All clones produced this way were then subc loned back into the wild-type L gene. Instead of sequencing the entire mutant DNA, only a region containing the mutation site

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12 between the restriction sites to be used fo r subcloning was sequen ced. Therefore that region, or insert, was cut out in a double digestion (Figure 2-1) and ligated back into the WT L gene at those sites to ensure that no other mutations were present (Table 2-3). Some of the subclones were then tested for th e silent restriction s ite and the clones were tested again by TNT for expression of full le ngth L protein, while ot hers were sent to sequencing to test for presence of the insert DNA subcloned into WT L DNA. Some of the clones were produced sole ly from subcloning. Once many of the mutant clones had been made it was easier to subclone one into anot her than to start from the beginning with the QuikChange Kit. This procedure was performed the same as described above, however, instead of using WT L DNA as the vector another mutant clone was used as the vector. For example, hr 1-0,1 was made by subcloning the insert hr 1-1 into the vector hr 1-0. This mutant was then sequen ced to confirm the presence of both vector and insert.

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13 Figure 2-1. The pBS-SK-L plasmid was used as the WT-L template for cloning through mutagenesis. The inserted mutations were sequenced and then subcloned by cutting out the mutation in a double digestion, using Bst BI and Sal I, Xma I and Bst BI, or Xba I and Bst BI, and ligating it back into WT-L. Table 2-1. Primers used to incorporate the mutations of hr 1 and hr 8. Primer Primer Sequence SM 577 CAAAAAGGAAAGAT GGGCCGACTCATTCAAAATTC SM 578 GAATTTTGAATGAGTCGGCCCATCTTTCCTTTTTG DH-1 CAAATCCTGGGGCCGTACAGTTATTGGGACAACTG SM 533 CAGTTGTCCCAATAA CTGTACGGCCCCAGGATTTG DH-2 GGAAGAATTTAGCA CCTGCTGAGCAATCC SM 534 GGATTGCTCAGCAGGTGCTAAATTCTTCC SM 538 GGAGACTTGGCCT CGAGAAAATCTACTC SM 535 GAGTAGATTTTCTCGAGGCCAAGTCTCC SM 617 CCTCCTATCCGACTAGTAA CCGTGATATGAGGGTGATTGTCAG SM 618 CTGACAATCACCCTCATA TCACGGTTACTAGTCGGATAGGAGG SM 580 GACACAAAGGCTAC CGACTGGAAAGAATTTC SM 581 GAAATTCTTTCCAGTCGGTAGCCTTTGTGTC

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14 Table 2-2. Mutagenic primers and incor porated silent restriction sites. For Mutagenesis: Sense AntisenseSilent Mutant* Template Primer Primer Restriction Site Hr8-0 pBS VSV-L SM 577 SM 578 HaeIII Hr8-1 pBS VSV-L DH-1 SM 533 BcgI Hr8-2 pBS VSV-L DH-2 SM 534 HaeII Hr8-3 pBS VSV-L SM 538 SM 535 XhoI Hr8-4 pBS VSV-L SM 617 SM 618 SpeI Hr8-1,2 hr8-1 DH-2 SM 534 HaeII Hr8-1,2,3 hr8-1,2 SM 538 SM 535 XhoI Hr8-1,2,3,4 hr8-1,2,3 SM 617 SM 618 SpeI Hr8-0,1,2,3 hr8-1,2,3 SM 577 SM 578 HaeIII Hr8-0,1,2,3,4 Hr1-0 pBS VSV-L SM 580 SM 581 BsrI Hr1-1 Made by Rich Hall Hr1-0,1 *Mutagenesis was performed using the primer s and a silent restri ction site was added. Hr 1-1 was made by Rich Hall, hr 8-0,1,2,3,4 and hr 1-0,1 were produced solely by subcloning.

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15 Table 2-3. Subcloning enzymes, vectors, and inserts. For Subcloning: Mutant Vector Insert Enzymes hr8-0 hr8-0 pBS VSV-L XbaI & BstBI hr8-1 pBS VSV-L hr8-1 SalI & BstBI hr8-2 pBS VSV-L hr8-2 SalI & BstBI hr8-3 pBS VSV-L hr8-3 SalI & BstBI hr8-4 pBS VSV-L hr8-4 SalI & BstBI hr8-1,2 pBS VSV-L Hr8-1,2 SalI & BstBI hr8-1,2,3 pBS VSV-L Hr8-1,2,3 SalI & BstBI hr8-1,2,3,4 pBS VSV-L hr8-1,2,3,4 SalI & BstBI hr8-0,1,2,3 hr8-0,1,2,3 pBS VSV-L XmaI & BstBI hr8-0,1,2,3,4 hr8-0 hr8-1,2,3,4 SalI & BstBI hr1-0 pBS VSV-L hr1-0 SalI & BstBI hr1-1 Made by Rich Hall hr1-0,1 hr1-0 Hr1-1 SalI & BstBI *Subcloning was performed by using two enzymes to cut the DNA and an insert was then ligated into the vector. Cells and Virus A549, baby hamster kidney (BHK), and HEp-2 cells all from ATCC were used to express VSV proteins from plasmids in vivo The cells were maintained using F11medium. For BHK cells it was supplem ented with 10% fetal bovine serum, 1% nonessential amino acids, 2% glutamine, 1% pe nicillin-streptomycin, and 0.35% glucose. The medium for A549 and HEp-2 cells was supplemented with 8% fetal bovine serum, 1% nonessential amino acids, 1% glutamine, 1% penicillin-streptomycin, and 1% sodium pyruvate. Cells were grown to ~80% conf luency on 35 mm dishes for infection and transfection. The vaccinia virus-T7 polymer ase recombinant and the derivative MVA-T7 virus were the gift of B. Moss (NIH).

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16 TNT Coupled Transcription/Tr anslation Reaction System The TNT Coupled Transcription/Translati on Reaction System (Promega) was used to verify that the mutant clones produced full length L proteins. Approximately 1ug of DNA was mixed with 6.25 ul TNT Rabbit Reticu locyte lysate, 0.5 ul TNT buffer, 0.25 ul T7 RNA polymerase, 0.25 ul amino acids minus leucine, 0.5 ul RNasin, and 4.25 ul Hleu. The reaction was mixed gently and inc ubated at 30C for 2 hours. The product was separated on a 7.5% SDS polyacrylamide gel next to wild-type L from WT L DNA to determine if the mutant was producing fu ll length L protein. The gel was washed, impregnated with PPO, dried, and incubate d with x-ray film at -80C overnight. In vivo Expression of L Mutant Proteins A549 cells were split 1:30 the day prio r to infection and grown to ~80% confluency in 35 mm dishes. The cells were then infected with VVT7, a vaccinia virus recombinant encoding T7 RNA polymerase, at an m.o.i. of 2.5 pfu/cell using F11 infection medium. The infected dishes were rocked for 1 hour at 37C. The infection medium was then aspirated and the cells were transfected with 2 ug VSV P and 2 ug VSV L (WT and mutants) plasmids in Opti-MEM and 3 ul lipofectamine per 1 ug of DNA for four hours at 37C. Four hours after transfection, the tr ansfection media was aspirated and 35S methionine overnight label (1 ug) media (2 ml) was added (100 uCi/ml or 35S methionine, 1 ml F11, 0.1 ml penicillin-streptomycin, 0.1ml glutamine, 0.1ml HEPES, pH 8.0, and 9 ml cysteine/methionine-free MEM). The follo wing day the media was aspirated and the cells were washed with cold PBS. The cells were scraped into a sterile microfuge tube with 100 ul of 1% NP40 lysis buffer using sterile rubber polic emen. The cells were then spun down for 30 min at 13,000 rpm at 4C. Th e supernatant (10 ul) was analyzed on a

PAGE 26

17 7.5% SDS polyacrylamide gel. The gel was wa shed, dried, and incubated with film at 80C overnight. CAT (Chloramphenicol A cetyltransferase) Assay The DI-CAT plasmid (Figure 2-2), constr ucted by Dr. Michael Baron, contains a gene encoding minus sense chloramphenicol ace tyltransferase flanked by the 3’ and 5’ termini of VSV genome RNA downstream of a T7 promoter. Cells are infected with MVA expressed T7 RNA polymerase which drives the synthesis of VSV polymerase complex plasmids (N, P, and L) as well as DI-CAT. N, P, and L mRNAs are then translated by the cellular machinery. DI-CAT is synthesized by the T7 polymerase to produce an antisense copy of the VSV mi nigenome with the minus sense CAT ORF between the VSV 5’ and 3’ termini. The ribozyme contained in the DI-CAT plasmid cleaves the 3’ end of the RNA after transc ription leaving only the CAT RNA which is inefficiently and nonspecifically encapsidate d. This creates a template for the viral polymerase to replicate the RNA to a positive sense minigenome. Then transcription of the template occurs to produce a functional mRNA (Figure 2-3). The mRNA can then be translated into protein (CAT) by the cellular translation machinery. A549 cells were split 1:30, BHK cells were cut 1:24, or HEp-2 cells were cut 1:24 the day prior to infection and grown to ~80% confluency in 35 mm dishes. The cells were then infected with MVA-T7, a m odified vaccinia virus expressing T7 RNA polymerase, at an m.o.i. of 2.5 pfu/cell using F11 infection medium. The infected dishes were rocked for 1 hour at 37C. The infection medium was then aspirated and the cells were transfected with plasmids containing 1 ug N, 0.3 ug L (WT or mutant), 0.5 ug P, 0.5 ug DI-CAT along with Opti-MEM and 3 ul of lipofectamine pe r 1 ug of DNA. The cells were transfected for 24 hours at either 34C, 37C, or 40C.

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18 Twenty four hours post-transfection, the tr ansfection media was aspirated and fresh supplemented F11 media was added. The following day the media was aspirated and rinsed with cold PBS. 200 ul of 0.25 M Tris-HCl, pH 7.8 and 0.5% Triton X-100 was added to the dishes and the cells were scrape d with sterile rubber policemen. The scraped cells were spun down in sterile microfuge tubes for 10 min at 13,000 rpm at 4C. The CAT-ELISA Kit (Roche) was used to test the abilities of the L mutants to direct replication and transcription compared to WT VSV L. This was done by ELISA for CAT protein. First, 50 ul of the supernatant was added to wells containi ng 150 ul of sample buffer (provided by the kit). Incubating th e wells for 1 hour at 37C allows the CAT protein to bind to the walls of the wells. The wells were washed five times with 200 ul of 10X Wash solution dilute d 1:10. Then, 200 ul of -CAT-DIG antibody (1:100 in sample buffer) was added to the wells. Af ter the second 1 hour incubation, the -CAT-DIG has bound to the proteins. Again, the wells are washed five times. Finally, 200 ul of -DIGPOD antibody (3:400 in sample buffer) was added to the wells. This binds to the -CATDIG during the 1 hour incubation. The wells are washed five more times and then 200 ul of POD substrate is added (Fi gure 2-4). The substrate turn s green in the presence of bound -DIG-POD. A plate reader determines th e ratio of protein present compared to mock and WT. All mutants were tested in triplicate in a minimum of two separate experiments and mock was subtracted from each sample. Therefore, each mutant has at least six results.

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19 Figure 2-2. Representation of the DI-CAT pl asmid used in the CAT assay to measure VSV RNA polymerase activity. Figure 2-3. Representation of the transfor mation of the CAT ORF into a functional mRNA.

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20 Figure 2-4. Representation of antibo dy binding in Roche CAT-ELISA kit.

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21 CHAPTER 3 RESULTS: HR 1 Clones The original hr 1 mutant contains two amino aci d changes from WT L, ASP in place of ASN at position 505 and VAL in place of ASP at position 1671. In order to examine the effect that each amino acid cha nge has on the function of the L protein, the mutations needed to be reproduced individually in the L protein. Therefore, each amino acid change was remade indi vidually and the original hr 1 mutant containing both mutations was also reconstructed. Hr 1-0 was constructed using the Strata gene QuikChange-XL Site-Directed Mutagenesis Kit described in the Material and Methods section. In summary, a PCR product was made where the WT or mutant L gene was used as the template and two complementary mutagenic primers created the desired mutation with a silent restriction site. If the silent restriction site, whic h was located near the desired mutation, was incorporated into the L gene the DNA was isol ated by a Qiagen Midi-prep or Maxi-prep. Then, a TNT coupled transcription/translati on (results below) was performed on the DNA to verify that it produced a full length L protein. Then a shor t region containing the mutation was sequenced to determine that the sequence was actually correct. That short region was subcloned back into the WT L gene via a double digest ion and ligation to ensure that no other mutations were present. Hr 1-1 had been made previously by Dr. Rich Hall in our laboratory. The combination hr 1-0,1 was made strictly by subcloning the hr 1-1 mutant region into hr 1-0.

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22 TNT Coupled Transcription/Translation Reaction Analysis of L Proteins The amino acid changes that were intr oduced through the Stratagene QuikChangeXL Site-Directed Mutagenesis Kit were not meant to truncate the L protein, but only to exchange one amino acid for another. Therefore, hr 1-0, which was produced via the Stratagene Kit, was tested by the TNT system described in Chapter 2 to verify that it produced full length L protein. Each DNA was mixed with the reagents and 3H leucine and incubated at 37C. The product was se parated on an SDS-PAGE and detected by autoradiography. Although synthesis of the WT L protein in Lane 1 failed, Lane 5 in Figure 3-1 shows that hr 1-0#22 is producing full length protein similar to hr 8-0, another mutant L protein which was us ed as a marker. The other hr 1-0 clone in Lane 4 had another mutation that tr uncated the protein. Hr 1-0 was then sequenced by UF’s DNA Sequencing Core to further confirm that only the correct amino acid had been incorporated into the L gene. Figure 3-1. TNT of hr 1-0 and other hr mutants. Analysis of the TNT product by SDSPAGE shows that hr 1-0 #22 is producing full length L-protein.

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23 In vivo Expression of L Mutants Not only is it important to ensure the L mutants produce full length L protein in a TNT reaction system, but it is also necessary for the L mutants to produce full length L protein in vivo The in vivo expression system described in Ch apter 2 was used to test the ability of all of the L mutant plasmids, wh ether produced via mutagenesis or subcloning, to produce full length L proteins in cells. Cells were infected with VVT7 and then transfected with VSV P and VSV L plasmids using lipofectamine. After four hours, the transfection media was aspirated and 35S methionine overnight label was added. The following day, the cells were rinsed and scrape d into a sterile microf uge tube. After the cells were spun down, and lyzed, the nuclei re moved and the supernatant was analyzed on an SDS-PAGE. Figure 3-2 compares th e synthesis of L protein produced by the mutants to the WT L protein. All of the hr 1 mutants are producing full length L protein at about the same level as compared to WT L. P protein cannot be determined due to cell background labeling. Figure 3-2. In vivo expression of hr 1 L-mutants. This SDS-PAGE gel shows that the mutants all produce full length L-pr otein compared to WT in an in vivo system.

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24 Analysis of the hr 1 L Mutants for Transcription and Replication The CAT-ELISA Kit (Roche) was used to te st the abilities of the L mutants to direct replication and transc ription from a CAT reporter mi nigenome. This was done by ELISA for the chloramphenicol acetyltransferase protein. As described in more detail in Chapter 2, the DI-CAT plasmid contains a gene encoding minus sense CAT downstream of a T7 promoter. Cells are infected with a modified vaccinia virus encoding a T7 RNA polymerase, which drives the synthesis of VS V N, P and L RNAs from plasmids as well as DI-CAT RNA. The N, P, and L mRNAs are then translated by the cellular machinery. DI-CAT is synthesized by the T7 polymerase to produce a minus sense CAT minigenome that is cleaved from the remaining R NA by a ribozyme contained in the DI-CAT transcript. This RNA is nonspecifically encap sidated by N proteins to create a template for the viral polymerase to replicate the R NA first to a positive sense and then to a negative sense minigenome. Then viral tran scription of the amplified negative sense template occurs to produce a functional CA T mRNA. The mRNA is translated into chloramphenicol acetyltransferase protein by the cellular translation machinery. The Roche kit measures the amount of translated chlorampheni col acetyltransferase produced by the L mutants as compared to WT L as a measure of RNA synt hesis. A CAT mRNA that is unmethylated during transcription will be inefficiently translated however, in this system vaccinia methyltransferases would me thylate any unmethylated CAT mRNA. So methylation activity of L is not being meas ured. All CAT assays summarized below were performed twice on each mutant in triplicate. The original hr 1 mutant has host restriction as well as temperature sensitivity. Therefore, CAT assays were performed at three different temperatures and in three separate cell lines. The permissive a nd nonpermissive temperatures are 34C, 40C

PAGE 34

25 respectively, while 37C represents a centr al point between the two where virus is normally grown. BHK cells are permissive while the HEp-2 and A549 cell lines are nonpermissive. To test for temperature sensitivity in nonpermissive cells, CAT assays were performed for the L mutants on HEp-2 cells at all three temperatures. Tables 3-1, 3-2, and 3-3 show the percent of CAT protein tran slated by the L mutants relative to that of WT L at 34C, 37C and 40C, respectively. In order to be considered temperature sensitive, the activity must drop by at least 80% at the hi gher temperature. At 34C (Table 3-1), the three mutants are relatively WT except for hr 1-1, which is 57% of WT L, indicating that 34C does not sign ificantly affect the activity of the L mutants. When the temperature is increased to 37C, hr 1-0 loses about 43% of its activity when compared to hr 1-0 at the nonpermissive temperature. But, hr 1-1 loses over 80% of its activity, suggesting that the hr 1-1 mutation makes L temperature se nsitive (Table 3-2). At 40C (Table 3-3), the hr 1-1 mutant still performs at only 20% of WT L thus further supporting the temperature sensitivity of th is protein. Interestingly, the hr 1-0,1 double mutant at 37C and 40C is not as reduced in viral RNA synthesis as hr 1-1 alone. Perhaps the mutation at amino acid 505 stabilizes the protein. While th e activity of the hr 1-0 protein is decreased at 37C compared to 34C, it appear s similar to WT L at 40C. It is unclear what the cause of this result is. These expe riments show that when the mutant proteins are made in these nonpermissive cells at 34C significant activity is obtained, suggesting that the nonpermissive host cell is not a de terminate of RNA synthesis. However, the hr 1-1 protein is temperature sensitive due to the single mutation at amino acid 1671.

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26 Table 3-1. CAT synthesis of hr 1 mutants in HEp-2 cells at 34C. HEp-2 Cells @34C Mutant Average* WT 100 hr1-0 126 hr1-1 57 hr1-0,1 100.5 *VVT7 infected HEp-2 cells we re transfected and incubated at 34C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. Table 3-2. CAT synthesis of hr 1 mutants in HEp-2 cells at 37C. HEp-2 Cells @37C Mutant Average* WT 100 hr1-0 54 hr1-1 7.55 hr1-0,1 29.7 *VVT7 infected HEp-2 cells we re transfected and incubated at 37C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. Table 3-3. CAT synthesis of hr 1 mutants in HEp-2 cells at 40C. HEp-2 Cells @40C Mutant Average* WT 100 hr1-0 119.5 hr1-1 22.5 hr1-0,1 48 *VVT7 infected HEp-2 cells we re transfected and incubated at 40C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. To further test the possible host range e ffects, CAT assays were also performed on A549 and BHK nonpermissive and permissive cell lines, respectively, at 37C (Tables 3-4 and 3-5). Data fairly similar to that obt ained in HEp-2 cells at 37C was obtained. It appears that L mutants synthesized in A549 ce lls are slightly more active than in HEp-2

PAGE 36

27 cells. Hr 1-1 again appears temperature sensitive regardless of the cell line. The basis for the host range of the mutant virus does not appear to be a lack of RNA synthesis. Table 3-4. CAT synthesis of hr 1 mutants in A549 cells at 37C. A549 Cells @37C Mutant Average* WT 100 hr1-0 50 hr1-1 28 hr1-0,1 56.5 *VVT7 infected A549 cells were transfected and incubated at 37C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. Table 3-5. CAT synthesis of hr 1 mutants in BHK cells at 37C. BHK Cells @37C Mutant Average* WT 100 hr1-0 ND hr1-1 20.5 hr1-0,1 ND *VVT7 infected BHK cells were transfected and incubated at 37C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. Dr. Valery Grdzelishvili et al. (in press) performed experiments on the hr 1 L mutants that were inserted into recombinant vesicular stomatitis virus to look at cap methylation. His findings are clearer and mo re precisely support th e result that both temperature sensitivity and host range phenotypes reside in the hr 1-1 mutation. Table 36 compares both host range and temperature sensitivity by plaque assays of the virus under different conditions. The second to last column shows the ratio of virus titers at 34C with respect to 40C in BHK cells. The increased temperature had a significant inhibitory effect in hr 1-1 virus and the original, as well as the reconstructed hr 1-0,1,

PAGE 37

28 viruses but not on hr 1-0 virus as suggested by the CAT assays above. Additionally, the ratio of virus titers in BHK and HEp-2 cells is shown in the last column. Again, the hr 11 virus and the original and reconstructed hr 1 mutant viruses are clearly deficient for growth in HEp-2 cells, although hr 1-0 is not. These results clearly show that both host range and temperature sensitiv ity lie in the same mutation, hr 1-1 at amino acid 1671 in L. Table 3-6. Comparative tite rs of recombinant VSV mutants in BHK and HEp-2 cell lines at 34C and 40C (Grdzel ishvili et al., In Press). Virus titer (PFU/ml) in BHK cells Virus titer (PFU/ml) in HEp-2 cells Virus 34C 40C 34C 40C Titer ratio 34C/40C in BHK Titer ratio 34C in BHK/HEp-2 Wt 2.6 x 109 6.0 x 1086.5 x 1078.0 x 1064.3 40.0 rHR1-0 2.5 x 109 1.0 x 1094.4 x 1078.5 x 1062.5 56.8 rHR1-1 4.2 x 108 < 103 < 103 < 103 > 105 > 105 rHR1-0,1 1.1 x 109 < 103 < 103 < 103 > 106 > 106 hr1 (Orig.) 2.5 x 109 1.5 x 104< 103 < 103 1.7 x 105 >106

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29 CHAPTER 4 RESULTS: HR 8 Clones The original hr 8 mutant contains five amino aci d changes from WT L, ALA in place of THR at position 148 ( hr 8-0) ILE in place of THR at position 1097 ( hr 8-1), ASP in place of ALA at position 1356 ( hr 8-2), SER in place of TYR at position 1375 ( hr 8-3), and ARG in place of GLY at position 1481 ( hr 8-4). In order to examine the effect that each amino acid change has on the function of the L protein, the mutations needed to be reproduced individually in the L protein. Therefore, each amino acid change was constructed individually or in combinations and the original hr 8 mutant containing all five mutations was also reconstructed. Two clones were made prior to the start of this project: hr 8-1 and hr 8-2. Eight more clones were successfully produced fo llowing the mutagenesis and subcloning methods described in Chapter 2. They are: hr 8-0, hr 8-3, hr 8-4, hr 8-1,2, hr 8-1,2,3, hr 81,2,3,4, and hr 8-0,1,2,3,4. Hr 8-0 and hr 8-0,1,2,3,4 were later determined to be the incorrect sequence. Therefore, they as well as hr 8-0,1,2,3 were remade by Sherin Smallwood in our laboratory. All subse quent tests proved th at the three new hr 8 mutants were correct (resu lts not shown). TNT Coupled Transcription/Translation Reaction Analysis of L Proteins All the clones produced via the Stratagene QuikChange-XL Site-Directed Kit were tested for full length protein by the TNT sy stem as the amino acid changes introduced were not meant to truncate the L protein. Those mutants are: hr 8-0, hr 8-3, hr 8-4, hr 8-1,2

PAGE 39

30 hr 8-1,2,3, hr 8-0,1,2,3, hr 8-1,2,3,4. Figures 4-1, 4-2, and 4-3 show the products of the TNTs analyzed by SDS-PAGE. Figure 4-1 indicates that each of the hr 8-1,2 and hr 8-3 clones are producing full length L prot ein relative to th e WT L control. In Figure 4-2, the subclones of hr 8-1,2 and hr 8-1,2,3 are shown and they are also both producing full length L protein. Figure 4-3 shows a mutant th at did not produce full length protein. Hr 8-4#2 is truncated and that mutant was discarded how ever the other clone is correct. Both hr 81,2,3,4 clones produced full length L proteins. Following the TNT reaction, clones that were producing full length L protein were then sequenced by UF’s DNA Sequencing Core. Figure 4-1. TNT of hr 8-1,2 and hr 8-3. Analysis of the TNT products by SDS-PAGE shows that hr 8-1,2 and hr 8-3 mutants are producing full length L-protein when compared with WT (control). Nu mbers 4 and 13 were used in future experiments.

PAGE 40

31 Figure 4-2. TNT of hr 8-1,2 and hr 8-1,2,3 subclones. Analysis of the TNT products by SDS-PAGE shows that the subclones of hr 8-1,2 and hr 8-1,2,3 mutants are producing full length L-protein. Figure 4-3. TNT of hr 8-4 and hr 8-1,2,3,4. Analysis of the TNT products by SDS-PAGE shows that hr 8-4 #1 and hr 8-1,2,3,4 are producing fu ll length L-protein. In vivo Expression of L Mutants All of the L mutants, whether produced via mutagenesis or subcloning must produce full length L protein in vivo The in vivo expression system described in Chapter 2 was used to test protein synthesis of the L mutants by radiolabeling infected, transfected cells. Figure 4-4 compares the L proteins produced by th e mutants to the WT L protein. All the hr 8 mutants are producing full length L protein. In other experiments

PAGE 41

32 (data not shown) reconstructed hr 8-0, hr 8-0,1,2,3 and hr 8-0,1,2,3,4 also gave full length L proteins in cells. Figure 4-4. In vivo expression of hr 8 L-mutants. This SDS-PAGE gel shows that the mutants all produce full length L-pr otein compared to WT in an in vivo system. Lanes from left to right: Mock, WT VSV, hr 8-0, hr 8-1, hr 8-2, hr 8-3, hr 8-4, hr 8-1,2, hr 8-1,2,3, hr 8-1,2,3,4, hr 8-0,1,2,3,4. Hr 8-0 and hr 8-0,1,2,3,4 were later determined to be incorrect sequences. Analysis of the hr 8 L Mutants for Transcription and Replication The CAT-ELISA Kit (Roche) was used to te st the abilities of the L mutants to direct replication and tran scription from a CAT reporter minigenome compared to WT VSV L. The CAT minigenome (described in mo re detail in Chapter 2) is constructed so that when it is transfected into cells along with N, P and L, chloramphenicol acetyltransferase protein is produced. The Ro che kit measures the amount of translated CAT produced as a measure of viral RNA synt hesis. All CAT assays summarized below were performed twice on each mutant in triplicate. Like the original hr 1 mutant virus, the original hr 8 mutant virus showed host restriction as well as temperature sensitivity. Therefore, CAT assays were performed at three different temperatures and in three separate cell lines. The permissive and

PAGE 42

33 nonpermissive temperatures are 34C and 40C, respectively, while 37C represents a central point between the two where virus is normally grown. BHK cells are permissive, while the HEp-2 and A549 cell lines are non-permissive. To test for temperature sensitivity, CAT assays were performed for the L mutants on HEp-2 cells at all three temperatures. Ta bles 4-1, 4-2, and 4-3 show the percent of CAT protein translated by the L mutants relati ve to that of WT L at 34C, 37C and 40C respectively. In order to be considered temperature sensitive, the activity of the L mutants must drop by at least 80% at the higher temperatures. At the permissive temperature of 34C (Table 4-1), most of the mutants are reduced somewhat but not below 50% of WT L. Hr 8-4 and hr 8-0,1,2,3,4 are more reduced to 43% and 34% respectively. Every mutant shows some e ffect on RNA synthesis at the permissive temperature. Surprisingly, as the temperat ure was increased to 37C and 40C (Tables 42 and 4-3), the mutants apparently become more heat resistant. Especially notable is hr 81,2 which is greatly stimulated to 600% of WT L. The r eason is unknown but it could be that WT L is actually less stable at the nonpermissive temperatures than the L mutants giving higher mutant values. In any case, th e results do not point to a specific mutation as being responsible for the temperature sensitivity of hr 8 virus.

PAGE 43

34 Table 4-1. CAT synthesis of hr 8 mutants in HEp-2 cells at 34C. HEp-2 Cells @34C Mutant Average* WT 100 hr8-0 61 hr8-1 71 hr8-2 85 hr8-3 50 hr8-4 43 hr8-1,2 ND hr8-1,2,3 ND hr8-1,2,3,4 69 hr8-0,1,2,3 52 hr8-0,1,2,3,4 34 *VVT7 infected HEp-2 cells we re transfected and incubated at 34C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. ND – Not Done. Table 4-2. CAT synthesis of hr 8 mutants in HEp-2 cells at 37C. HEp-2 Cells @37C Mutant Average* WT 100 hr8-0 ND hr8-1 ND hr8-2 ND hr8-3 100.5 hr8-4 77.5 hr8-1,2 ND hr8-1,2,3 79 hr8-1,2,3,4 66 hr8-0,1,2,3 147.5 hr8-0,1,2,3,4 ND *VVT7 infected HEp-2 cells we re transfected and incubated at 37C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. ND – Not Done.

PAGE 44

35 Table 4-3. CAT synthesis of hr 8 mutants in HEp-2 cells at 40C. HEp-2 Cells @40C Mutant Average* WT 100 Hr8-0 63 Hr8-1 146 Hr8-2 280 Hr8-3 48.5 Hr8-4 130.5 Hr8-1,2 602.5 Hr8-1,2,3 73 Hr8-1,2,3,4 108.5 Hr8-0,1,2,3 135.5 Hr8-0,1,2,3,4 85 *VVT7 infected HEp-2 cells we re transfected and incubated at 40C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. To test for host range effects, CAT a ssays were performed for the L mutants on A549 and BHK cells at 37C (Tables 4-4 and 4-5). When experiments were conducted on A549 cells (Table 4-4), the results were largely similar to HEp-2 cells, however hr 80,1,2,3 and hr 8-0,1,2,3,4 are not included in these resu lts as they were being remade. Because hr 8-4 had somewhat decreased activity in Table 4-2, it was tested along with hr 8-0,1,2,3,4 in the permissive cell line, BHK (T able 4-5). As expected, the mutants were also more active in the permissive cel l line at 37C like in HEp-2 cells at 37C. Thus the cell line appeared to play no role in the activity of the L protein. Even at a permissive temperature each of the mutant s seemed to have some effect on RNA synthesis and increased temperatures increa sed rather than decreased activity. As indicated in Chapter 3, the vaccinia virus T7 polymerase that infected the cells probably methylated the mRNAs allowing CAT to be mo re efficiently translated. Clearly the

PAGE 45

36 minigenome assays with expres sion of mutant L proteins are not predictive of functions seen in virus. Table 4-4. CAT synthesis of hr 8 mutants in A549 cells at 37C. A549 Cells @37C Mutant Average* WT 100 hr8-0 115.5 hr8-1 171.5 hr8-2 107.3 hr8-3 81.5 hr8-4 131 hr8-1,2 153 hr8-1,2,3 74.5 hr8-1,2,3,4 38 hr8-0,1,2,3 ND hr8-0,1,2,3,4 ND *VVT7 infected A549 cells were transfected and incubated at 37C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. ND – Not Done Table 4-5. CAT synthesis of hr 8 mutants in BHK cells at 37C. BHK Cells @37C Mutant Average* WT 100 hr8-4 100 hr8-0,1,2,3,4 41.5 *VVT7 infected BHK cells were transfected and incubated at 37C. Six to nine CAT assay experiments were averaged and calcu lated against WT assuming 100% activity with less than 15% variation. As with the hr 1 mutants, Valery Grdzelishvili performed experiments on the hr 8 L mutants that were inserted into recombinan t vesicular stomatitis virus. His findings clearly indicate that both the temperature sens itivity and the host ra nge phenotypes of the hr 8 virus reside in the hr 8-4 mutation. The second to last column of Table 4-6 shows that any recombinant virus containing the -4 mutatio n is significantly less active at 40C than

PAGE 46

37 at 34C while all the other hr 8 mutations are like WT at the nonpermissive temperature. Additionally, the ratio of titers for each virus in BHK and HEp-2 cells is shown in the last column. Again, hr 8-4, hr 8-1,2,3,4, hr 8-0,1,2,3,4 and the original hr 8 mutant are deficient in HEp-2 cells relative to BHK ce lls, while the other mutants are not. These results clearly show that both host range and temperature sensitivity phenotypes lie in the same mutation, hr 8-4 with a change from GLY to ARG at amino acid 1481. Table 4-6 Comparative ti ters of recombinant VSV mutants in BHK and HEp-2 cell lines at 34C and 40C. Virus titer (PFU/ml) in BHK cells Virus titer (PFU/ml) in HEp-2 cells Virus 34C 40C 34C 40C Titer ratio 34C/40C in BHK Titer ratio 34C in BHK/HEp-2 Wt 2.6 x 109 6.0 x 1086.5 x 1078.0 x 1064.3 40.0 rHR8-0 8.0 x 108 7.0 x 1081.5 x 1071.5 x 1061.1 53.0 rHR8-1 6.0 x 109 6.0 x 1091.1 x 1084.5 x 1061.0 54.5 rHR8-2 2.0 x 108 2.0 x 1085.5 x 1061.2 x 1061.0 36.4 rHR8-3 3.5 x 109 2.0 x 1091.1 x 1088.0 x 1061.8 31.8 rHR8-4 3.0 x 109 < 103 6.0 x 104< 103 > 3.0 x 106 5.0 x 104 rHR8-0,1,2,3 3.5 x 109 2.0 x 1093.2 x 1085.0 x 1071.7 10.9 rHR8-1,2,3,4 3.2 x 108 < 103 3.0 x 103< 103 > 105 > 105 rHR8-0,1,2,3,4 2.0 x 109 2.0 x 1044.0 x 103< 103 2.0 x 105 5.0 x 105 hr8 (Orig.) 4.0 x 108 < 103 < 103 < 103 > 4.0 x 105 >4.0 x 105

PAGE 47

38 CHAPTER 5 DISCUSSION The overall focus of my research was to determine which amino acid change or combination of changes in the hr 1 and hr 8 L mutant viruses were responsible for the host range and temperature sensitivity phenotypes. The minigenome CAT assays that were performed on the constructed L mutants whic h measured the effect of temperature and host cells on RNA synthesis by the expressed pr oteins were not completely predictive. When comparing the CAT assay re sults to other data obtained in our laboratory, it is clear that assaying the L protein in cell culture is not the same as assaying the protein in a virus infection. The CAT assays suggested that the hr 1 mutation responsible for temperature sensitivity is the replacement of VAL for A SP at position 1671 regard less of the cell line in which it was expressed. The results of Gr dzelishvili et al (J. Virology, 2005, in press) do show that hr 1-1 is, in fact, the mutation responsi ble for temperature sensitivity. His plaque assay experiments on recombinant VSV, which had the hr 1 L mutations (Figure 3-6), show that the virus titer ratio at 34 C/40C is five logs greater in recombinant hr 1-1 and hr 1-0,1 and the original hr 1 mutant indicating that a ll L proteins containing the hr 1-1 mutation are temperature sensitive. While the CAT assay results were predic tive of temperature sensitivity in hr 1, it was unclear which amino acid change was affecting host restriction as all the hr 1 mutants had fairly similar activities when compared to WT L regardless of the cell line in which they were expressed. In nonpermissive cells such as HEp-2 and A549, the original L

PAGE 48

39 mutant does not methylate mRNA, however, mR NA is methylated in permissive cells, like BHK (Horikami et al., 1984 and Horikami and Moyer, 1982). This is because viral transcription occurs in the cytoplasm of th e cell. The nucleus of the cell contains methyltransferase enzymes that are leaked out into the cytoplasm in BHK cells, but not in nonpermissive cells. Therefore the hr 1 mutation responsible for unmethylated mRNAs is rescued by the permissive cells eliminating the appearance of a defect while the nonpermissive cells are unable to rescue methylation and th e amount of translated CAT protein would be decreased. Because the CAT assay requires cells to be infected with VVT7, which encodes its own methyltransf erase enzymes, it is likely that the recombinant vaccinia virus rescued the methylation in nonpermissive cells eliminating the appearance of a defect. Thus, this assa y cannot measure methyla tion affects in the L protein. Grdzelishvili et al conducted plaque assay e xperiments to test for host restriction (J. Viro logy, 2005, in press) and concluded that hr 1-1 is once again the responsible mutation. The ratios of virus ti ters in BHK and HEp-2 cells are five logs greater in hr 1-1, hr 1-0,1, and the original hr 1 mutant indicating that L proteins containing the hr 1-1 mutation are host restricted as non-pe rmissive cells are unable to rescue the methylation defect. Additional experiments have been conduc ted by Grdzelishvili to prove that hr 1-1 is in fact causing a methylation defect. He demonstrated in our laboratory that the hr 1-1 mutation does not have a signi ficant effect on mRNA synthesi s in virus. Additionally, his results showed that this L mutation di d not affect capping of viral mRNA during in vitro transcription. Howeve r, when he conducted in vitro transcription by detergentactivated purified viruses using a labele d methyl donor (AdoMet), it was clear that hr 1-1,

PAGE 49

40 hr 1-0,1 and the original hr 1 mutants were not being methylated at all (results not shown). Further, when the cap structures of WT and hr 1-0 mutants were analyzed by digesting the [3H]AdoMet labeled RNA with P1 nuclease to release the cap, the digestion products were shown to be 7mGpppAm and GpppAm indicating that the non-mutant virus mRNAs (WT and hr 1-0) were being methylated at the cap structure, confirming previous results with the original hr 1 virus (Horikami et al., 1984). The hr 1 mutation at position 1671 is located in conserved Domain VI of the L protein. Figure 5-1 indicates the location of Domain VI and also shows the sequence of several L proteins of other negative stra nded RNA viruses with similar conserved regions. Similar conserved regions in cellula r and viral methyltransferases have been proven to be the S-adenosylmethionine binding site and mutations in this area are known to abolish the activities of two met hylating enzymes, mRNA guanine-N7 cap methyltransferase and nucl eoside-2’-O cap methyltran sferase (Luongo et al., 1998, Mao and Shuman, 1996, Wang and Shuman, 1997). Based on this data, we propose that the mutation at amino acid 1671 abolishes A doMet binding and therefore all cap methylation.

PAGE 50

41 Figure 5-1. A) Conserved domains in VSV and the location of the hr 1-1 mutation. B) The same conserved regions in known S-adenosylmethionine binding sites that are responsible for guanine-N7 cap methyltransferases. C) The same conserved regions in known S-adenos ylmethionine binding sites that are responsible for nucleoside-2’O cap methyltransferases. In the case of hr 8, the data on the analysis of vi ral RNA synthesis were unclear and unexpected. When testing temperatur e sensitivity, none of the mutants were significantly decreased due to an increase in temperature. Surprisingly, hr 8-2 and hr 81,2 appear to be WT at the permissive temper ature but were actually more stable relative to WT L at 40C. The hr 8-2 mutation is found in the nonconserved region between Domains V and VI at position 1356. Perh aps this mutation changes the overall conformation of the protein making it more stab le. Host restriction results were equally inconclusive when analyzing the CAT assay results of hr 8. Like hr 1, the VVT7 required

PAGE 51

42 for the CAT assays most likely rescued the methylation defect thus eliminating the appearance of a mutation. It was impossible to predict which mutant was affected by temperature and the host cells by reviewing the CAT assay results. However, Grdzelishvili et al. (manuscript in preparation) have now shown by plaque assays that hr 8-4 at position 1488 is the single mutation responsible for both temperature sensit ivity and host restriction. The ratios of virus titers at 34C/40C and in BHK/ HEp-2 are both five log greater for hr 8-4, hr 81,2,3,4, hr 8-0,1,2,3,4 and the original hr 8 mutant indicating that any mutant containing the hr 8-4 mutation is both temperatur e sensitive and host restricted. Similar experiments that were conducted on hr 1 are in progress to further test for the methylation abilities of hr 8-4. Grdzelishvili’s initial results indicate that hr 8-4 is not temperature sensitive for RNA synthesis but only for methylation. He has found that methylation is not abolished, as in hr 1, but it is decreased by a pproximately 95%. It was originally thought that hr 8 caused a defect spec ifically in the 7mG methyltransferase. Horikami and Moyer (1982) found that hr 8 mRNA could be mono-methylated at the penultimate base or not methylat ed at all. However, some current results indicate that hr 8 can be methylated at the 7m G position as well as at the 2’ O penultimate base at very low levels. The hr 8-4 mutation at position 1488 is loca ted in the nonconserved region between Domains V and VI of the L protein. This change causes a phenotype of limited methylation. Therefore, position 1488 is not part of the SAM bi nding site, as partial methylation can be achieved. It is possible that position 1488 is part of the catalytic site for one or both of the methylating enzymes. Hr 8-4 could also change the overall

PAGE 52

43 conformation of the protein thus decreasing its ability to me thylate. Another option is that hr 8-4 is part of a catalytic site for one enzyme and changes the overall conformation of the protein affecting the other methylat ion event. Grdzelishvili’s results, when completed, will help to determine the functi on that amino acid 1488 plays in methylation. Additionally, site-directed mutagenesis w ill be conducted in the future on the area between amino acids 1400 and 1671 to furt her analyze the potential role of the nonconserved region between Doma ins V and VI on methylation.

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44 LIST OF REFERENCES Abraham G, Rhodes DP, Banerjee AK. The 5’ terminal structure of the methylated mRNA synthesized in vitro by ve sicular stomatitis virus. Cell 1975;5:51-58. Ball LA, White CN. Order of transcription of genes of vesicular stomatitis virus. Proc Natl Acad Sci USA 1976;73:442-446. Blumberg BM, Giorgi C, Kolakofsky D. N protein of vesicular stomatitis virus selectively encapsidates leader RNA in vitro. Cell 1983;32:559-567. Blumberg BM, Kolakofsky D. Intracellular vesicular stomatitis virus leader RNAs are found in nucleocapsid structures. Journal of Virology 1981;40:568-576. Buchholz CJ, Spehner D, Drillien R, et al The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly. Journal of Virology 1993;67:5803-5812. Canter DM, Jackson RL, Perraul t J. Faithful and efficient in vitro reconstitution of vesicular stomatitis virus transcription using plasmid-encoded L and P proteins. Virology 1993;194:518-529. Cevik B, Smallwood S, Moyer SA. The L-L oligomerization domain resides at the very N-terminus of the Sendai virus L RNA polymerase protein. Virology 2003;313:525-536. Cortese CK, Feller JA, Moyer SA. Mutati ons in Domain V of the Sendai Virus L Polymerase Protein Uncouple Transcription and Replication and Differentially Affect Replication in Vitro and in Vivo Virology 2000;277:387-396. Curran J, Pelet T, Kolakofsky D. An acidic activation-like domain of the Sendai virus P protein is required for RNA synthesis and encapsidation. Virology 1994;202:875-884. Feller JA, Smallwood S, Horikami SM, Moye r SA. Mutations in Conserved Domains IV and VI of the Large (L) Subunit of the Sendai Virus RNA Polymerase Give a Spectrum of Defective RNA Synthesis Phenotypes. Virology 2000;269:426-439. Grdzelishvili VZ, Smallwood S, Tower D, Hall RL, Hunt DM, Moyer SA. A single amino acid change in the L polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. Journal of Virology (In Press).

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45 Gringas AC, Raught B, Sonenberg N. eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 1999;68:913-963. Hercyk N, Horikami SM, Moyer SA. The vesicular stomatitis virus L protein possesses the mRNA methyl transferase activities. Virology 1988;163:222-225. Horikami SM, De Ferra F, Moyer SA. Characterization of the infections of permissive and nonpermissive cells by hos t range mutants of vesicular stomatitis virus defective in RNA methylation. Virology 1984;138:1-15 Horikami SM, Moyer SA. Host range mutants of vesicular stomatitis virus defective in in vitro RNA methylation. Proc Natl Acad Sci 1982;79:7694-7698. Iverson LE, Rose JK. Localized attenua tion and discontinuous synthesis during vesicular stomatitis virus transcription. Cell 1981;23:447-484. Jin H, Elliott RM. Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies. J. Gen. Virol. 1992;73:2235-2244. Kolakofsky, D, Bidal S, Curran J. Pa ramyxovirus RNA synthesis and P gene expression. In The Paramyxoviruses. Ki ngsbury DW, Ed. Plenum Press, New York, 1991;215-233. Luongo CL, Contreras CM, Farsetta DL, Nibert ML. Binding site for S-adenosyl-Lmethionine in a central region of mammalia n reovirus lambda2 protein. Evidence for activities in mRNA cap methylation. Journal of Biol. Chem. 1998;273:23773-23780. Mao X, Shuman S. Vaccinia virus mRNA (guanine-7-)methyltransferase: mutational effects on cap methylation a nd AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site. Biochemistry 1996;35:6900-6910. Moyer SA, Banerjee AK. Messenger RNA speci es synthesized in vitro by the virionassociated RNA polymerase of vesicular stomatitis virus. Cell 1975;4:37-43. Parks GD, Ward CD, Lamb RA. Molecular cl oning of the NP and L genes of simian virus 5: Identification of highly conserved domains in Paramyxovirus NP and L proteins. Virus Res 1992;22:259-279. Poch O, Blumberg BM, Bougueleret L, and Tordo N. Sequence comparison of five polymerases (L proteins) of unsegmented ne gative-strand RNA viruse s: Theorhetical assignment of functional domains. Journal of Virology 1990;71:1153-1162. Robbins SJ, Bussell RH. Structural phosphopr oteins associated with purified measles virions and cytoplasmic nucleocapsids. Intervirology 1979;12:96-102.

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46 Rose JK, Whitt MA. Rhabdoviridae: The Viruses and Their Replication. In Fundamental Virology, 4th Edition. Lippincott Williams and Wilkins, Philadephia, 2001;665-668. Ryan KW, Portner A. Separate domains of Sendai virus P protein are required for binding to viral nucleocapsids. Virology 1990;174:515-521. Schnell MJ, Conzelmann KK. Polymerase activity of in vitro mutated rabies virus L protein. Virology 1995;214:522-530. Schuber M, Keene JD, Herman RC, Lazzarini RA. Site of the vesicular stomatitis virus genome specifying polyadenylation at the end of the L gene mRNA. J Virol 1980;34:550-559. Sidhu MS, Menonna JP, Cook SD, Dowling PC and Udem SA. Canine distemper virus L gene: Sequence and comp arison with related viruses. Virology 1993;193:5065. Simpson RW, Obijeski JF. Conditional lethal mu tants of vesicular stomatitis virus. I. Phenotypic characterization of single and double mutants exhibiting host restriction and temperature sensitivity. Virology 1974;57:357-368. Slack MS, Easton AJ. Characterization of the interaction of the human respiratory syncytial virus phosphoprotein and nucleocapsid protein using the two-hybrid system. Virus Res 1998;55:167-176. Sleat DE, Banerjee AK. Transcriptiona l activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase. Virology 1993;67:13341339. Smallwood S, Cevik B, Moyer SA. Intragenic Complementation and Oligomerization of the L Subunit of the Sendai Virus RNA Polymerase. Virology 2002;304:235-245. Smallwood S, Easson CD, Feller JA, Horikami SM, Moyer SA. Mutations in conserved domain II of the large (L) s ubunit of the Sendai virus RNA polymerase abolish RNA synthesis. Virology 1999;262:375-383. Stephens EB, Compans RW, Earl P, Moss B. Surface expression of viral glycoproteins is polarized in epithelial cells infected with reco mbinant vaccinia viral vectors. EMBO J 1986;5:237-245. Stillman EA, Whitt MA. Transcript initiation and 5’-end modification. J Virol 1999;73:7199-7209. Tarbouriech N, Curran J, Ebel C, et al. On the domain structure and the polymerization state of the sendai virus P protein. Virology 2000;266:99-10.

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47 Wang SP, Shuman S. Structure-f unction analysis of the mRNA cap methyltransferase of Saccharomyces cervisiae. Journal of Biol Chem 1997;272:14683-14689.

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48 BIOGRAPHICAL SKETCH Dallas Lauren Tower was born in Mobile, Alabama, in 1979. In 1990, she moved to Fairfax, Iowa. After graduating from Cedar Rapids Prairie High School in 1997, Dallas attended Iowa State University. In December, 2000, she received her Bachelor of Science degree in biology and started worki ng for Integrated DNA Technologies, Inc. Dallas moved to Gainesville, Florida in 2002 to pursue graduate education. She received her Master of Science degree in medical sciences and her Master of Business Administration degree in May, 2005.


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Title: The Effect of Putative Vesicular Stomatitis Virus Methyltransferase Mutants on Transcription and Replication
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Permanent Link: http://ufdc.ufl.edu/UFE0010088/00001

Material Information

Title: The Effect of Putative Vesicular Stomatitis Virus Methyltransferase Mutants on Transcription and Replication
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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THE EFFECT OF PUTATIVE VESICULAR STOMATITIS VIRUS
METHYLTRANSFERASE MUTANTS ON TRANSCRIPTION AND REPLICATION















By

DALLAS L. TOWER


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005


































Copyright 2005

by

Dallas L. Tower















ACKNOWLEDGMENTS

First and foremost, I would like to thank my parents for supporting me in all my

endeavors and instilling in me the strong desire to accomplish all of my goals. I owe a

tremendous debt of gratitude to my parents for their encouragement and assistance

throughout my academic career. I thank my lab members, Sherin Smallwood, Valery

Grdzelishvili, and Dorothy Smith, for their assistance, guidance, and friendship

throughout my tenure at the University of Florida. I would like to thank my committee

members, Sue Moyer, Donna Duckworth, and Richard Condit, for their advice on my

project. My advisor, Sue Moyer, has been an incredible mentor to me over the past three

years. I thank her for her patience and guidance. She has instilled in me a deep respect

for scientific researchers and I cannot thank her enough for the education I have received

under her counseling.















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TA BLES .................................................................... ............ .. vi

LIST OF FIGURE S ......... ..................................... ........... vii

A B STR A C T ... .................... ............................................ ... ....... ....... viii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

G general Background ............................................... ......... .. .............. 1
Transcription .................................... ................... ............. .......... 2
R e p lic atio n ............................. .............................................................. ............... 3
A ssem bly and R release ........................ .............. .......... .................. .3
P protein s ............................................................. . 4
M P ro te in .................................................................................. 4
G P protein ................................................................................... . 4
N P protein ................................................ 4
P Protein ............................................... 5
L Protein ......................................................................................................... 5
Capping and Methylating Mechanism.............................................8
Project B background ..................................................................... .................. 8

2 M A TERIALS AN D M ETH OD S ......................................................................... 11

C lo n e s ................................................................ ........................................... 1 1
C ells and V irus ......................... ..... ..... ................................................ 15
TNT Coupled Transcription/Translation Reaction System ................... ...........16
In vivo Expression of L Mutant Proteins ............................................................16
CAT (Chloramphenicol Acetyltransferase) Assay .....................................17

3 R E SU L T S: H R 1......................................................................................21

Clones ........................... ........... ... .... ........ ................21
TNT Coupled Transcription/Translation Reaction Analysis of L Proteins ..............22
In vivo Expression of L Mutants .................................... ...............23
Analysis of the hrl L Mutants for Transcription and Replication ..............................23










4 R E S U L T S : H R 8 ................................................................................. .................... 2 9

Clones ............................ ........... ... .... ........ ................29
TNT Coupled Transcription/Translation Reaction Analysis of L Proteins .............29
In vivo Expression of L M utants .................................................. ............ ............... 31
Analysis of the hr8 L Mutants for Transcription and Replication ...........................32

5 D ISCU SSION .......... .......................................................................................... 38

L IST O F R E F E R E N C E S ............................................................................ ............... 44

B IO G R A PH IC A L SK E T C H ...................................................................... ..................48












































v
















LIST OF TABLES


Table pge

1-2 N om enclature of hrl m utants. ........................................ ......................................10

1-3 N om enclature of hr8 m utants. ........................................ ......................................10

2-1 Primers used to incorporate the mutations ofhrl and hr8. .....................................13

2-2 Mutagenic primers and incorporated silent restriction sites................... ............14

2-3 Subcloning enzymes, vectors, and inserts. ...................................... ............... 15

3-1 CAT synthesis of hrl mutants in HEp-2 cells at 340C..........................................26

3-2 CAT synthesis of hrl mutants in HEp-2 cells at 370C..................... ..................26

3-3 CAT synthesis of hrl mutants in HEp-2 cells at 400C.............................................26

3-4 CAT synthesis of hrl mutants in A549 cells at 37C. .............................................27

3-5 CAT synthesis of hrl mutants in BHK cells at 37C ................................................27

3-6 Comparative titers of recombinant VSV hrl mutants in BHK and HEp-2 cell lines
340C and 40C...... ............................................... 28

4-1 CAT synthesis ofhr8 mutants in HEp-2 cells at 340C..........................................34

4-2 CAT synthesis ofhr8 mutants in HEp-2 cells at 370C................................... 34

4-3 CAT synthesis of hr8 mutants in HEp-2 cells at 400C..............................................35

4-4 CAT synthesis ofhr8 mutants in A549 cells at 37C. .........................................36

4-5 CAT synthesis ofhr8 mutants in BHK cells at 370C..........................................36

4-6 Comparative titers of recombinant VSV hr8 mutants in BHK and HEp-2 cell lines
3 4 0C an d 4 0 C ................................................. ................ 3 7
















LIST OF FIGURES

Figure page

1-1 Schematic of the VSV gene order........... ............................. ...... ...................3

1-2 Conserved regions in Sendai L protein.. .............................................................7

2-1 The pBS-SK-L plasmid used as the WT-L template for cloning through
m utag en esis. ......................................................... ................ 13

2-2 Representation of the DI-CAT. ............................................................................ 19

2-3 Representation of the transformation of the CAT ORF into a functional mRNA. ..19

2-4 Representation of antibody binding in Roche CAT-ELISA kit.............................20

3-1 TNT of hrl-0 and other hr mutants............................................................ ........ 22

3-2 In vivo expression of hrl L-mutants.. ................................................. ............... 23

4-1 TN T of hr8-1,2 and hr8-3................................... ............................................... 30

4-2 TNT of hr8-1,2 and hr8-1,2,3 subclones. ............................................................31

4-3 TNT of hr8-4 and hr8-1,2,3,4. ............ .......................................... ....................3 1

4-4 In vivo expression of hr8 L-mutants. ............. ............................... ...............32

5-1 Conserved domains in VSV and the location of the hrl-1 mutation .......................41



















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

THE EFFECT OF PUTATIVE VESICULAR STOMATITIS VIRUS
METHYLTRANSFERASE MUTANTS ON TRANSCRIPTION AND REPLICATION

By

Dallas L. Tower

May 2005

Chair: Sue A. Moyer
Major Department: Molecular Genetics and Microbiology

The vesicular stomatitis virus (VSV) mRNAs are methylated at the guanine-N7 and

2'-O-adenosine positions. The RNA-dependent RNA polymerase synthesizes the viral

mRNAs which are modified at their 3' and 5' ends by polyadenylation, capping, and

methylation. Two host range mutants, hrl and hr8, isolated by Simpson and Obijeski are

defective in methylation. WT mRNA caps are dimethylated by 7-methylguanosine and

2'-O-methyltransferase yielding the cap structure 7mGpppAmpAp. The cap on an

mRNA from hrl has no methylation (GpppApAp) and the cap on an mRNA from hr8

was thought be 2'-O-mono-methylated or not methylated at all. Unmethylated mRNAs,

like those produced by hrl and hr8, are deficient in protein synthesis. Further, hrl and

hr8 are host restricted as well as temperature sensitive. The N, P, and L proteins that

form the polymerase and the respective genes of the hrl and hr8 mutants were sequenced









and compared to WT N, P and L genes. Hrl has two amino acid substitutions differing

from WT in the L gene and hr8 has five amino acid substitutions, also found in the L

gene. In this study, the hrl and hr8 mutations were introduced separately, together, and

combination into the L gene. The effects on the VSV L mutant and WT L proteins were

studied in CAT minigenome assays. The assays were conducted in permissive and

nonpermissive cell lines as well as permissive and nonpermissive temperatures. The

second amino acid change at position 1671 in the hrl mutant appears to be responsible

for the temperature sensitivity. The data on the hr8 mutant proved to be inconclusive.

Further studies performed in the Moyer lab confirm that amino acid 1671 is responsible

for temperature sensitivity as well as the host restriction. Additionally, amino acid

position 1481 is responsible for both host restriction and temperature sensitivity. Position

1671 may play a critical role within the S-adenosylmethionine binding domain and

position 1481 could possibly be part of the catalytic site for 7mG methyltransferase.














CHAPTER 1
INTRODUCTION

General Background

Vesicular Stomatitis Virus (VSV) is a Vesiculovirus found in the order

Mononegavirales. A number of viruses causing human diseases are contained in this

order, including measles, mumps, rabies, Ebola, Marburg, Boma, Hendra, and Nipah.

More specifically VSV is found in the family Rhabdoviridae, which also includes rabies.

Over 200 rhabdoviruses have been identified. Vesiculovirus is one of six genera in the

Rhabdoviridae family.

VSV contains two serotypes, Indiana and New Jersey, both of which infect a broad

range of insects and mammals. The virus is present in North and South America and

relatives of the virus can be found in Africa and Asia. Symptoms of the disease resemble

foot and mouth disease with vesicular lesions on the tongue, gums, teats and hooves in

livestock. Headache, nausea, malaise and general influenza-like symptoms are also

possible. The virus is spread through vesicular fluid and saliva; it can also be transmitted

by infected flies and mosquitoes. VSV can infect humans causing a mild illness.

VSV, like rabies, is a bullet shaped virus. It is approximately 180 nm long by 75

nm wide. The virus is enveloped with G-protein spikes on the surface. It has a single-

stranded negative sense RNA genome of 11,200 nucleotides. RNA that is negative sense

cannot be translated immediately after entry into the cell. Therefore, an antigenomic

intermediate must be produced by the RNA-dependent RNA polymerase, which comes

packaged in the virus, in order to make a viable mRNA.









Transcription

Before transcription can occur, the virus must attach to a receptor on a host which

initiates the infection. Next, the virion is endocytosed by the host cell and the

ribonucleoprotein (RNP) core with the attached RNA-dependent RNA polymerase

(RdRp) is released into the cytoplasm. The RNP with N-encapsidated, negative strand

RNA serves as the template for transcription. The RdRp, which consists of two viral

subunits: the phosphoprotein (P) and the large protein (L), transcribes the template to

produce (+) strand mRNAs which are modified at their 3' and 5' ends by

polyadenylation, capping, and methylation, respectively.

The viral genes are transcribed sequentially in VSV, producing leader RNA and

then the capped and methylated mRNAs with a gene order of 5'-N-P-M-G-L-3' (Ball and

White, 1976) (Figure 1-1). VSV polymerase pauses between each gene and then begins

synthesizing the next downstream gene (Iverson and Rose, 1981). Possibly, this is to

generate the poly(A) tail on the mRNA.

Additionally, the polymerase is responsible for capping the mRNA (See Capping

and Methylating Mechanism). It is thought that conserved sequences preceding and

within each gene contain the capping and reinitiation signals (Stillman and Whitt, 1999).

Cap methylation, involving the methyl donor S-adenosyl-L-methionine (AdoMet), is

essential for translation of viral mRNAs but not mandatory in viral mRNA synthesis

(Gingras et al., 1999, Horikami et al., 1984, Horikami and Moyer 1982).













N P M G L






Figure 1-1. Schematic of the VSV gene order. Image taken from Rose and Whitt (2001).


Replication

Replication of VSV occurs after the synthesis of mRNAs and their translation.

Replication requires ongoing protein synthesis. Genomic replication cannot occur until N

protein is synthesized. Additionally, the P and L proteins, which form the polymerase

catalyzing transcription, are also responsible for replication. Sufficient encapsidation of

leader RNA by the N protein signals the polymerase to ignore mRNA initiation and

termination signals and consequently produces full length positive-sense genome RNA

(Blumberg et al., 1983). Genome RNA synthesis is always coupled to its encapsidation

by N. The encapsidated positive-stranded genome RNA is replicated again to produce

the encapsidated genomic negative-stranded RNA.

Assembly and Release

Encapsidated RNA (RNP) is now ready for assembly and release. The matrix

protein (M) condenses the RNP and packages it along with the RNA-dependent RNA

polymerase. The glycoprotein (G), which produces the spikes on the surface of the

virus, is transported through the secretary pathways of the cell to the outer cell plasma

membrane. In VSV, this is typically on the basolateral surface of cells (Stephens et al.,

1986). The M protein brings the assembled RNP and viral polymerase complex to the









cell plasma membrane through an interaction of the G and M proteins. As the virus buds

out from the cell membrane, the glycoprotein spikes become the new exterior of the

virus.

Proteins

M Protein

The M protein, or matrix protein, is derived from the third mRNA to be transcribed

from the viral genome. M is the smallest protein of the five VSV proteins. It condenses

the nucleocapsid for packaging, disrupts the cytoskeleton, and aids the budding of the

virus from the cell.

G Protein

The G protein, or glycoprotein, is derived from the fourth mRNA to be transcribed

from the viral genome. It is a membrane protein responsible for the coat of spikes (-400)

on the virion membrane. The G protein enhances the efficiency of budding 10- to 30-

fold (Rose and Whitt, 2001). G protein attaches the virus to the susceptible cell and is the

protein to which neutralizing antibody is directed.

N Protein

The N protein, or nucleocapsid protein, is derived from the first mRNA to be

transcribed from the viral genome. N is a cytoplasmic protein with several functions. N

is responsible for packaging the RNA into a compact core that serves as the template for

transcription and replication. The P-L polymerase complex binds to N on the RNP

during transcription and replication and N interacts with the M protein during virus

assembly. As mentioned previously, N is thought to control the rates of transcription and

replication as well as the transition between transcription and replication (Blumberg and

Kolakofsky, 1981). The N protein probably has two domains: the highly conserved









(-80%) N-terminus and the poorly conserved (-20%) C-terminus (Parks et al., 1992).

Deletion experiments by Buchholz et al. (1993) suggest that the highly conserved N-

terminal region is required for RNA binding and nucleocapsid assembly. The P protein

of the polymerase complex binds the C-terminus of N in RNP.

P Protein

The P protein, or phosphoprotein, is derived from the second mRNA to be

transcribed from the viral genome. The P protein was initially thought to be a

homotrimer, but more recent studies indicate that it is actually a tetramer (Tarbouriech, et

al., 2000). P works in combination with the L protein in forming the RNA-dependent

RNA polymerase. It alone is not responsible for enzymatic activity, but probably helps

fold the L protein for activity (Kolakofsky et al., 1991). Additionally, P binds to N and

preserves the soluble state of N in the cytoplasm until RNA genome synthesis requires N

for encapsidation (Robbins and Bussell, 1979).

The coiled-coil oligomerization regions of the P protein in the C-terminal end are

necessary for the interaction between P and L (Ryan and Portner, 1990). P protein

oligomerization occurs in many viruses, such as respiroviruses, morbilliviruses,

Rubulavirus, and RSV (Slack and Easton, 1998). Therefore, oligomerization is thought

to be a general characteristic of the protein.

The N-terminus contains a small region that is essential for RNA encapsidation.

The specific function of the region is unknown. However deletion of residues 1 to 78 of

the P protein hinders RNA synthesis and encapsidation (Curran et al., 1994).

L Protein

The L protein, named for its large size, is derived from the fifth mRNA to be

transcribed from the viral genome. Like P, the L protein is also an oligomer (Smallwood









et al., 2002, Cevik et al., 2003). Containing more than 2100 amino acids in a single

polypeptide chain, it is the largest of the five VSV proteins. The L protein is the catalytic

subunit of the RNA-dependent RNA polymerase. It is a multifunctional protein involved

in transcription, replication, mRNA capping, methylation, and polyadenylation (Abraham

et al., 1975, Hercyk et al., 1988, Schubert et al., 1980).

Due to its multifunctional characteristics, it was believed that the L protein

consisted of several functional regions. Further experiments confirmed this hypothesis.

When L protein sequences from several (-) sense RNA viruses were compared, six

conserved regions were found and named Domains I to VI (Poch et al., 1990, Sidhu et al.,

1993) (Figure 1-2). Domains II, III and VI contain conserved motifs that appear to be

essential for RNA synthesis. Domain II contains a charged RNA binding motif

(Smallwood et al., 1999). A template recognition/phosphodiester bond-forming motif,

mandatory for VSV RNA synthesis, has been mapped to Domain III (Jin and Elliot,

1992, Sleat and Banerjee 1993, Schnell and Conzelman, 1995). In 1993, Canter et al.

showed that a purine binding element essential for polymerization could be eliminated

with one deletion in Domain VI of VSV L.

Additional studies by Feller et al., 2000, showed that when mutations are inserted

in Domains IV and/or Domain VI a broad range of RNA synthesis defects occur. For

example, Sendai virus L protein becomes heat sensitive and can no longer transcribe

effectively. Some of the mutations resulted in a decrease in le+ RNA synthesis and even

a lack of RNA synthesis initiation. However, mutations in Domain IV and VI of the L

protein in Sendai virus did not result in the inability of L to bind P. Mutations in Domain










V also gave multiple defects in RNA synthesis, however the majority of the mutants were

defective in replication but not transcription (Cortese et al., 2000).

The original thought was that each of the six conserved regions of the L protein

corresponded to a separate activity (Poch et al., 1990, Sidhu et al., 1993). However,

when Smallwood et al. (2002) and Cevik et al. (2003) used site-directed mutagenesis to

create mutants in each of the domains, they noticed a commonality of RNA synthesis

defects between each of the domains. Therefore, similar phenotypes can be attained

through multiple mutations across the domains suggesting multiple independent domains.

This was supported by the finding that two L proteins that are deficient in RNA synthesis

alone can complement each other and restore RNA synthesis in vitro, and this

complementation is dependent on cotranslation of the L mutants, suggesting that L is an

oligomer in the polymerase complex (Smallwood et al., 2002).

3" emirl of
(-) genorne
le NP P/C/V M F HN le
4 I = I I I _r .





Jr_1l II
o -








Figure 1-2. Conserved regions in Sendai L protein. Amino acid positions are indicated
above the beginning of the conserved region. Image taken from Feller et al. (2000).











Capping and Methylating Mechanism

Abraham et al. (1975) suggest that capping is mediated by the polymerase

complex. But, so far no mutations located in the L protein have affected capping.

However, defects in viruses with a methyltransferase deficiency have been mapped to the

L protein (Hercyk et al., 1988). Cellular mRNA caps are formed by essentially three

enzymatic steps. First, RNA triphosphatase removes the y phosphate from the 5' end of

the nascent RNA. Then, guanylyltransferase adds a GMP in a 5'ppp5' linkage to the

nascent RNA. Lastly, methyl groups are added at G and the penultimate base forming

the 7mG and 2'0 base. However, 5' capping in non-segmented negative strand (NNS)

RNA viruses is different. The a and 0 phosphates in the GpppA phosphate bridge are

derived from a GDP donor (Abraham et al., 1975) which is added to GMP terminated

RNA. Then, the 5' terminus is methylated by guanine-N7-methyltransferase and

nucleoside-2'-O-methyltransferase activities of L (Moyer and Banerjee, 1975) to yield

m7GpppAmpNpNpNp.

Project Background

In the 1970's, Simpson and Obijeski were chemically mutagenizing VSV to

determine functional and structural properties of the viral genus (Simpson and Obijeski,

1974). Since VSV normally has such a broad host range, they attempted to isolate

mutants that exhibited host restriction (hr). VSV hr mutants share the same structural

proteins as WT VSV. Many of the mutants were host restricted in cells of human origin,

like HEp-2, or HeLa cells, but not in chicken embryo fibroblasts or hamster cells. Hrl,

for example, had deficient protein synthesis in nonpermissive cells. Some of the hr

mutants appeared to be temperature sensitive as well.









Normal viral mRNAs that are synthesized in vitro or in vivo are polyadenylated at

the 3' end and guanylated and methylated at the 5' end. However, further studies on the

hr mutants have shown that hrl is totally defective and hr8 is partially defective in vitro

in methylating the mRNA (Horikami and Moyer, 1982). A WT mRNA cap structure is

presumed to be methylated by two activities, 7-methylguanosine and 2'-O-

methyltransferase in L, yielding a dimethylated mRNA: 7mGpppAmpAp. The cap on an

mRNA from hrl has no methylation, GpppApAp, and the hr8 mRNA cap can be 2'O-

mono-methylated or not methylated at all. 7mG methylated mRNAs are required for

protein synthesis in mammalian cells, so unmethylated mRNAs give the deficient protein

synthesis described by Obijeski and Simpson (Simpson and Obijeski, 1974, Horikami

and Moyer, 1982).

The L protein was shown to possess the methyltransferase activities (Hercyk et al.,

1988) and sequencing of the VSV L, N, and P genes has shown that these hr mutations

are in fact in the L protein. Hrl has two amino acid changes from WT L and hr8 has five

changes (Table 1-1). The goal of my work is to determine which one amino acid change,

or combination of changes, is actually responsible for the host restriction and temperature

sensitivity of the viruses by measuring the effect each mutation in L has on transcription

and replication when compared to WT L. Each amino acid change has been constructed

separately in the WT L gene, and named as shown in Tables 1-2 and 1-3. Additionally,

several combinations of changes have also been made in the WT L gene. The original L

mutants of hrl and hr8 have also been reproduced by the combination of all the

mutations.









Table 1-1. Amino acid changes of hrl and hr8 from Simpson WT.

Amino Acid Simpson WT hrl hr8
148 THR ALA
505 ASN ASP
1097 THR ILE
1356 ALA ASP
1375 TYR SER
1481 GLY ARG
1671 ASP VAL

Table 1-2. Nomenclature of hrl mutants.

Nomenclature Amino Acid Simpson WT hrl*
hrl-0 505 ASN ASP
hrl-1 1671 ASP VAL


*The original hrl mutant contains both amino

Table 1-3. Nomenclature of hr8 mutants.


acid changes from


Simpson WT.


Nomenclature Amino Acid Simpson WT hr8*
hr8-0 148 THR ALA
hr8-1 1097 THR ILE
hr8-2 1356 ALA ASP
hr8-3 1375 TYR SER
hr8-4 1481 GLY ARG

*The original hr8 mutant contains all five mutations. Each individual mutation has a
name that corresponds with its amino acid change from Simpson WT.














CHAPTER 2
MATERIALS AND METHODS

Clones

In general, all the clones were produced by making a PCR product where the WT

VSV L gene or sequential hr mutant L genes were used as the template and two

complementary mutagenic primers (Table 2-1) with a silent restriction site created the

desired mutation. The pBS-SK-L plasmid (Figure 2-1) was used as the WT VSV L

template. The pBS-SK-L plasmid, as well as the pBS-N and pBS-P plasmids, was kindly

provided by J. Rose. The Stratagene QuikChangeXL Site-directed Mutagenesis Kit was

used to direct the PCR. The PCR product was digested with DpnI and then transformed

into the ultracompetent cells provided by the kit. The cells were plated on LB-Amp+

plates and colonies were then screened by PCR with primers bracketing the mutation site

and that PCR product was digested with an enzyme that corresponded to the introduced

silent restriction site. The silent restriction site was designed so as not to change the

amino acid sequence, but allow a cut site near the desired mutation to suggest that the

mutation was also present (Table 2-2). Colonies that contained the cut site were grown

up, and the DNA was isolated by a Qiagen Midi-prep or Maxi-prep. A TNT coupled

transcription/translation (see below) was performed on the DNA to make sure that it

produced a full length protein and the DNA was sequenced by UF's DNA Sequencing

Core to determine that it was actually correct.

All clones produced this way were then subcloned back into the wild-type L gene.

Instead of sequencing the entire mutant DNA, only a region containing the mutation site









between the restriction sites to be used for subcloning was sequenced. Therefore that

region, or insert, was cut out in a double digestion (Figure 2-1) and ligated back into the

WT L gene at those sites to ensure that no other mutations were present (Table 2-3).

Some of the subclones were then tested for the silent restriction site and the clones were

tested again by TNT for expression of full length L protein, while others were sent to

sequencing to test for presence of the insert DNA subcloned into WT L DNA.

Some of the clones were produced solely from subcloning. Once many of the

mutant clones had been made it was easier to subclone one into another than to start from

the beginning with the QuikChange Kit. This procedure was performed the same as

described above, however, instead of using WT L DNA as the vector another mutant

clone was used as the vector. For example, hrl-O,1 was made by subcloning the insert

hrl-1 into the vector hrl-O. This mutant was then sequenced to confirm the presence of

both vector and insert.











hrl-O


hr8-O


-I-


hr8-1 hr8-2 hr8-3


VSV


BstBI Sail


hr8-4


l X l
Xmal Xbal


pBS-SK-L
9425 bp


Figure 2-1. The pBS-SK-L plasmid was used as the WT-L template for cloning through
mutagenesis. The inserted mutations were sequenced and then subcloned by cutting out
the mutation in a double digestion, using BstBI and Sal, Xmal and BstBI, or Xbal and
BstBI, and lighting it back into WT-L.

Table 2-1. Primers used to incorporate the mutations of hrl and hr8.

Primer Primer Sequence
SM 577 CAAAAAGGAAAGATGGGCCGACTCATTCAAAATTC
SM 578 GAATTTTGAATGAGTCGGCCCATCTTTCCTTTTTG
DH-1 CAAATCCTGGGGCCGTACAGTTATTGGGACAACTG
SM 533 CAGTTGTCCCAATAACTGTACGGCCCCAGGATTTG
DH-2 GGAAGAATTTAGCACCTGCTGAGCAATCC
SM 534 GGATTGCTCAGCAGGTGCTAAATTCTTCC
SM 538 GGAGACTTGGCCTCGAGAAAATCTACTC
SM 535 GAGTAGATTTTCTCGAGGCCAAGTCTCC
SM 617 CCTCCTATCCGACTAGTAACCGTGATATGAGGGTGATTGTCAG
SM 618 CTGACAATCACCCTCATATCACGGTTACTAGTCGGATAGGAGG
SM 580 GACACAAAGGCTACCGACTGGAAAGAATTTC
SM 581 GAAATTCTTTCCAGTCGGTAGCCTTTGTGTC


hrl-1









Table 2-2. Mutagenic primers and incorporated silent restriction sites.

For Mutagenesis:
Sense Antisense Silent
Mutant* Template Primer Primer Restriction Site
Hr8-0 pBS VSV-L SM 577 SM 578 HaeIII
Hr8-1 pBS VSV-L DH-1 SM 533 BcgI
Hr8-2 pBS VSV-L DH-2 SM 534 HaeII
Hr8-3 pBS VSV-L SM 538 SM 535 Xhol
Hr8-4 pBS VSV-L SM 617 SM 618 Spel
Hr8-1,2 hr8-1 DH-2 SM 534 HaeII
Hr8-1,2,3 hr8-1,2 SM 538 SM 535 Xhol
Hr8-1,2,3,4 hr8-1,2,3 SM 617 SM 618 Spel
Hr8-0,1,2,3 hr8-1,2,3 SM 577 SM 578 HaeIII
Hr8-0,1,2,3,4
Hrl-0 pBS VSV-L SM 580 SM 581 BsrI
Hrl-1 Made by Rich Hall
Hrl-0,1

*Mutagenesis was performed using the primers and a silent restriction site was added.
Hrl-1 was made by Rich Hall, hr8-0,1,2,3,4 and hrl-0,1 were produced solely by
subcloning.









Table 2-3. Subcloning enzymes, vectors, and inserts.

For Subcloning:
Mutant Vector Insert Enzymes
hr8-0 hr8-0 pBS VSV-L XbaI & BstBI
hr8-1 pBS VSV-L hr8-1 Sail & BstBI
hr8-2 pBS VSV-L hr8-2 Sail & BstBI
hr8-3 pBS VSV-L hr8-3 Sail & BstBI
hr8-4 pBS VSV-L hr8-4 Sail & BstBI
hr8-1,2 pBS VSV-L Hr8-1,2 Sail & BstBI
hr8-1,2,3 pBS VSV-L Hr8-1,2,3 Sail & BstBI
hr8-1,2,3,4 pBS VSV-L hr8-1,2,3,4 Sail & BstBI
hr8-0,1,2,3 hr8-0,1,2,3 pBS VSV-L XmaI & BstBI
hr8-0,1,2,3,4 hr8-0 hr8-1,2,3,4 Sail & BstBI
hrl-0 pBS VSV-L hrl-0 Sail & BstBI
hrl-1 Made by Rich Hall
hrl-0,1 hrl-0 Hrl-1 Sail & BstBI

*Subcloning was performed by using two enzymes to cut the DNA and an insert was then
ligated into the vector.


Cells and Virus

A549, baby hamster kidney (BHK), and HEp-2 cells all from ATCC were used to

express VSV proteins from plasmids in vivo. The cells were maintained using

Fl1 medium. For BHK cells it was supplemented with 10% fetal bovine serum, 1%

nonessential amino acids, 2% glutamine, 1% penicillin-streptomycin, and 0.35% glucose.

The medium for A549 and HEp-2 cells was supplemented with 8% fetal bovine serum,

1% nonessential amino acids, 1% glutamine, 1% penicillin-streptomycin, and 1% sodium

pyruvate. Cells were grown to -80% confluency on 35 mm dishes for infection and

transfection. The vaccinia virus-T7 polymerase recombinant and the derivative MVA-T7

virus were the gift of B. Moss (NIH).









TNT Coupled Transcription/Translation Reaction System

The TNT Coupled Transcription/Translation Reaction System (Promega) was used

to verify that the mutant clones produced full length L proteins. Approximately lug of

DNA was mixed with 6.25 ul TNT Rabbit Reticulocyte lysate, 0.5 ul TNT buffer, 0.25 ul

T7 RNA polymerase, 0.25 ul amino acids minus leucine, 0.5 ul RNasin, and 4.25 ul 3H-

leu. The reaction was mixed gently and incubated at 300C for 2 hours. The product was

separated on a 7.5% SDS polyacrylamide gel next to wild-type L from WT L DNA to

determine if the mutant was producing full length L protein. The gel was washed,

impregnated with PPO, dried, and incubated with x-ray film at -800C overnight.

In vivo Expression of L Mutant Proteins

A549 cells were split 1:30 the day prior to infection and grown to -80%

confluency in 35 mm dishes. The cells were then infected with VVT7, a vaccinia virus

recombinant encoding T7 RNA polymerase, at an m.o.i. of 2.5 pfu/cell using Fl 1

infection medium. The infected dishes were rocked for 1 hour at 370C. The infection

medium was then aspirated and the cells were transfected with 2 ug VSV P and 2 ug

VSV L (WT and mutants) plasmids in Opti-MEM and 3 ul lipofectamine per 1 ug of

DNA for four hours at 370C.

Four hours after transfection, the transfection media was aspirated and 35S

methionine overnight label (1 ug) media (2 ml) was added (100 uCi/ml or 35S methionine,

1 ml F11, 0.1 ml penicillin-streptomycin, 0.lml glutamine, 0.lml HEPES, pH 8.0, and 9

ml cysteine/methionine-free MEM). The following day the media was aspirated and the

cells were washed with cold PBS. The cells were scraped into a sterile microfuge tube

with 100 ul of 1% NP40 lysis buffer using sterile rubber policemen. The cells were then

spun down for 30 min at 13,000 rpm at 40C. The supernatant (10 ul) was analyzed on a









7.5% SDS polyacrylamide gel. The gel was washed, dried, and incubated with film at -

800C overnight.

CAT (Chloramphenicol Acetyltransferase) Assay

The DI-CAT plasmid (Figure 2-2), constructed by Dr. Michael Baron, contains a

gene encoding minus sense chloramphenicol acetyltransferase flanked by the 3' and 5'

termini of VSV genome RNA downstream of a T7 promoter. Cells are infected with

MVA expressed T7 RNA polymerase which drives the synthesis of VSV polymerase

complex plasmids (N, P, and L) as well as DI-CAT. N, P, and L mRNAs are then

translated by the cellular machinery. DI-CAT is synthesized by the T7 polymerase to

produce an antisense copy of the VSV minigenome with the minus sense CAT ORF

between the VSV 5' and 3' termini. The ribozyme contained in the DI-CAT plasmid

cleaves the 3' end of the RNA after transcription leaving only the CAT RNA which is

inefficiently and nonspecifically encapsidated. This creates a template for the viral

polymerase to replicate the RNA to a positive sense minigenome. Then transcription of

the template occurs to produce a functional mRNA (Figure 2-3). The mRNA can then be

translated into protein (CAT) by the cellular translation machinery.

A549 cells were split 1:30, BHK cells were cut 1:24, or HEp-2 cells were cut 1:24

the day prior to infection and grown to -80% confluency in 35 mm dishes. The cells

were then infected with MVA-T7, a modified vaccinia virus expressing T7 RNA

polymerase, at an m.o.i. of 2.5 pfu/cell using F 11 infection medium. The infected dishes

were rocked for 1 hour at 370C. The infection medium was then aspirated and the cells

were transfected with plasmids containing 1 ug N, 0.3 ug L (WT or mutant), 0.5 ug P, 0.5

ug DI-CAT along with Opti-MEM and 3 ul of lipofectamine per 1 ug of DNA. The cells

were transfected for 24 hours at either 340C, 370C, or 400C.









Twenty four hours post-transfection, the transfection media was aspirated and fresh

supplemented F 11 media was added. The following day the media was aspirated and

rinsed with cold PBS. 200 ul of 0.25 M Tris-HC1, pH 7.8 and 0.5% Triton X-100 was

added to the dishes and the cells were scraped with sterile rubber policemen. The scraped

cells were spun down in sterile microfuge tubes for 10 min at 13,000 rpm at 40C. The

CAT-ELISA Kit (Roche) was used to test the abilities of the L mutants to direct

replication and transcription compared to WT VSV L. This was done by ELISA for CAT

protein. First, 50 ul of the supernatant was added to wells containing 150 ul of sample

buffer (provided by the kit). Incubating the wells for 1 hour at 370C allows the CAT

protein to bind to the walls of the wells. The wells were washed five times with 200 ul of

10X Wash solution diluted 1:10. Then, 200 ul of a-CAT-DIG antibody (1:100 in sample

buffer) was added to the wells. After the second 1 hour incubation, the a-CAT-DIG has

bound to the proteins. Again, the wells are washed five times. Finally, 200 ul of a-DIG-

POD antibody (3:400 in sample buffer) was added to the wells. This binds to the a-CAT-

DIG during the 1 hour incubation. The wells are washed five more times and then 200 ul

of POD substrate is added (Figure 2-4). The substrate turns green in the presence of

bound a-DIG-POD. A plate reader determines the ratio of protein present compared to

mock and WT. All mutants were tested in triplicate in a minimum of two separate

experiments and mock was subtracted from each sample. Therefore, each mutant has at

least six results.






19





DI-CAT PLASMID MAP


Il1 II I 660 S b,-r7--
TO TO a turn CAT ORF --- lrm.


T7 PN a, polymerase,
a ribozyme cleavage


3' cleaved RNA fragments +' NOT


antisense CAT ORF
! f


NOT MV:CAT chimeric


Figure 2-2. Representation of the DI-CAT plasmid used in the CAT assay to measure
VSV RNA polymerase activity.


NCT


antisense CAT ORF


NCT


SREPLICATN BY P AND L


NOT sense CAT ORF NcT

|/ REPLICATION BY P AND L
NOT -ti-sense CAT ORF NCOT

NCT antisense CAT ORF NCT
| ......... ,"'


(-Genomic, -CAT)





(+ Genomic. +CAT)




(-Genomic. -CAT)


TRANSCRIPTION BY P AND L


+ mRNA


Figure 2-3. Representation of the transformation of the CAT ORF into a functional
mRNA.


pBSKS(+








20








o o

o o00 o o
0 0



anti-CAT- CAT ant-CAT- ant-DIG- ABTS"
coated sample/ DIG PD substrate
MP standard Fab-fragmerrt

Figure 2-4. Representation of antibody binding in Roche CAT-ELISA kit.














CHAPTER 3
RESULTS: HR1

Clones

The original hrl mutant contains two amino acid changes from WT L, ASP in

place of ASN at position 505 and VAL in place of ASP at position 1671. In order to

examine the effect that each amino acid change has on the function of the L protein, the

mutations needed to be reproduced individually in the L protein. Therefore, each amino

acid change was remade individually and the original hrl mutant containing both

mutations was also reconstructed.

Hrl-0 was constructed using the Stratagene QuikChange-XL Site-Directed

Mutagenesis Kit described in the Material and Methods section. In summary, a PCR

product was made where the WT or mutant L gene was used as the template and two

complementary mutagenic primers created the desired mutation with a silent restriction

site. If the silent restriction site, which was located near the desired mutation, was

incorporated into the L gene the DNA was isolated by a Qiagen Midi-prep or Maxi-prep.

Then, a TNT coupled transcription/translation (results below) was performed on the DNA

to verify that it produced a full length L protein. Then a short region containing the

mutation was sequenced to determine that the sequence was actually correct. That short

region was subcloned back into the WT L gene via a double digestion and ligation to

ensure that no other mutations were present. Hrl-1 had been made previously by Dr.

Rich Hall in our laboratory. The combination hrl-0,1 was made strictly by subcloning the

hrl-1 mutant region into hrl-0.









TNT Coupled Transcription/Translation Reaction Analysis of L Proteins

The amino acid changes that were introduced through the Stratagene QuikChange-

XL Site-Directed Mutagenesis Kit were not meant to truncate the L protein, but only to

exchange one amino acid for another. Therefore, hrl-0, which was produced via the

Stratagene Kit, was tested by the TNT system described in Chapter 2 to verify that it

produced full length L protein. Each DNA was mixed with the reagents and 3H leucine

and incubated at 370C. The product was separated on an SDS-PAGE and detected by

autoradiography. Although synthesis of the WT L protein in Lane 1 failed, Lane 5 in

Figure 3-1 shows that hrl-0#22 is producing full length protein similar to hr8-0, another

mutant L protein which was used as a marker. The other hrl-0 clone in Lane 4 had

another mutation that truncated the protein. Hrl-0 was then sequenced by UF's DNA

Sequencing Core to further confirm that only the correct amino acid had been

incorporated into the L gene.





6 v -









Figure 3-1. TNT of hrl-0 and other hr mutants. Analysis of the TNT product by SDS-
PAGE shows that hrl-0 #22 is producing full length L-protein.











In vivo Expression of L Mutants

Not only is it important to ensure the L mutants produce full length L protein in a

TNT reaction system, but it is also necessary for the L mutants to produce full length L

protein in vivo. The in vivo expression system described in Chapter 2 was used to test the

ability of all of the L mutant plasmids, whether produced via mutagenesis or subcloning,

to produce full length L proteins in cells. Cells were infected with VVT7 and then

transfected with VSV P and VSV L plasmids using lipofectamine. After four hours, the

transfection media was aspirated and 35S methionine overnight label was added. The

following day, the cells were rinsed and scraped into a sterile microfuge tube. After the

cells were spun down, and lyzed, the nuclei removed and the supernatant was analyzed

on an SDS-PAGE. Figure 3-2 compares the synthesis of L protein produced by the

mutants to the WT L protein. All of the hrl mutants are producing full length L protein

at about the same level as compared to WT L. P protein cannot be determined due to cell

background labeling.








<- L






Figure 3-2. In vivo expression of hrl L-mutants. This SDS-PAGE gel shows that the
mutants all produce full length L-protein compared to WT in an in vivo system.









Analysis of the hrl L Mutants for Transcription and Replication

The CAT-ELISA Kit (Roche) was used to test the abilities of the L mutants to

direct replication and transcription from a CAT reporter minigenome. This was done by

ELISA for the chloramphenicol acetyltransferase protein. As described in more detail in

Chapter 2, the DI-CAT plasmid contains a gene encoding minus sense CAT downstream

of a T7 promoter. Cells are infected with a modified vaccinia virus encoding a T7 RNA

polymerase, which drives the synthesis of VSV N, P and L RNAs from plasmids as well

as DI-CAT RNA. The N, P, and L mRNAs are then translated by the cellular machinery.

DI-CAT is synthesized by the T7 polymerase to produce a minus sense CAT minigenome

that is cleaved from the remaining RNA by a ribozyme contained in the DI-CAT

transcript. This RNA is nonspecifically encapsidated by N proteins to create a template

for the viral polymerase to replicate the RNA first to a positive sense and then to a

negative sense minigenome. Then viral transcription of the amplified negative sense

template occurs to produce a functional CAT mRNA. The mRNA is translated into

chloramphenicol acetyltransferase protein by the cellular translation machinery. The

Roche kit measures the amount of translated chloramphenicol acetyltransferase produced

by the L mutants as compared to WT L as a measure of RNA synthesis. A CAT mRNA

that is unmethylated during transcription will be inefficiently translated however, in this

system vaccinia methyltransferases would methylate any unmethylated CAT mRNA. So

methylation activity of L is not being measured. All CAT assays summarized below

were performed twice on each mutant in triplicate.

The original hrl mutant has host restriction as well as temperature sensitivity.

Therefore, CAT assays were performed at three different temperatures and in three

separate cell lines. The permissive and nonpermissive temperatures are 340C, 40C









respectively, while 370C represents a central point between the two where virus is

normally grown. BHK cells are permissive, while the HEp-2 and A549 cell lines are

nonpermissive.

To test for temperature sensitivity in nonpermissive cells, CAT assays were

performed for the L mutants on HEp-2 cells at all three temperatures. Tables 3-1, 3-2,

and 3-3 show the percent of CAT protein translated by the L mutants relative to that of

WT L at 340C, 370C and 400C, respectively. In order to be considered temperature

sensitive, the activity must drop by at least 80% at the higher temperature. At 34C

(Table 3-1), the three mutants are relatively WT except for hrl-1, which is 57% of WT L,

indicating that 340C does not significantly affect the activity of the L mutants. When the

temperature is increased to 370C, hrl-0 loses about 43% of its activity when compared to

hrl-0 at the nonpermissive temperature. But, hrl-1 loses over 80% of its activity,

suggesting that the hrl-1 mutation makes L temperature sensitive (Table 3-2). At 40C

(Table 3-3), the hrl-1 mutant still performs at only 20% of WT L thus further supporting

the temperature sensitivity of this protein. Interestingly, the hrl-0,1 double mutant at

37C and 40C is not as reduced in viral RNA synthesis as hrl-1 alone. Perhaps the

mutation at amino acid 505 stabilizes the protein. While the activity of the hrl-0 protein

is decreased at 370C compared to 340C, it appears similar to WT L at 400C. It is unclear

what the cause of this result is. These experiments show that when the mutant proteins

are made in these nonpermissive cells at 340C significant activity is obtained, suggesting

that the nonpermissive host cell is not a determinate of RNA synthesis. However, the

hrl-1 protein is temperature sensitive due to the single mutation at amino acid 1671.









Table 3-1. CAT synthesis of hrl mutants in HEp-2 cells at 340C.

HEp-2 Cells @340C
Mutant Average*
WT 100
hrl-0 126
hrl-1 57
hrl-0,1 100.5

*VVT7 infected HEp-2 cells were transfected and incubated at 340C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation.

Table 3-2. CAT synthesis ofhrl mutants in HEp-2 cells at 370C.

HEp-2 Cells @370C
Mutant Average*
WT 100
hrl-0 54
hrl-1 7.55
hrl-0,1 29.7

*VVT7 infected HEp-2 cells were transfected and incubated at 370C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation.

Table 3-3. CAT synthesis ofhrl mutants in HEp-2 cells at 400C.

HEp-2 Cells @400C
Mutant Average*
WT 100
hrl-0 119.5
hrl-1 22.5
hrl-0,1 48

*VVT7 infected HEp-2 cells were transfected and incubated at 400C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation.

To further test the possible host range effects, CAT assays were also performed

on A549 and BHK nonpermissive and permissive cell lines, respectively, at 370C (Tables

3-4 and 3-5). Data fairly similar to that obtained in HEp-2 cells at 370C was obtained. It

appears that L mutants synthesized in A549 cells are slightly more active than in HEp-2









cells. Hrl-1 again appears temperature sensitive regardless of the cell line. The basis for

the host range of the mutant virus does not appear to be a lack of RNA synthesis.

Table 3-4. CAT synthesis ofhrl mutants in A549 cells at 370C.

A549 Cells @370C
Mutant Average*
WT 100
hrl-0 50
hrl-1 28
hrl-0,1 56.5

*VVT7 infected A549 cells were transfected and incubated at 370C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation.

Table 3-5. CAT synthesis ofhrl mutants in BHK cells at 370C.

BHK Cells @370C
Mutant Average*
WT 100
hrl-0 ND
hrl-1 20.5
hrl-0,1 ND

*VVT7 infected BHK cells were transfected and incubated at 370C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation.

Dr. Valery Grdzelishvili et al. (in press) performed experiments on the hrl L

mutants that were inserted into recombinant vesicular stomatitis virus to look at cap

methylation. His findings are clearer and more precisely support the result that both

temperature sensitivity and host range phenotypes reside in the hrl-1 mutation. Table 3-

6 compares both host range and temperature sensitivity by plaque assays of the virus

under different conditions. The second to last column shows the ratio of virus titers at

340C with respect to 400C in BHK cells. The increased temperature had a significant

inhibitory effect in hrl-1 virus and the original, as well as the reconstructed hrl-0,1,









viruses but not on hrl-0 virus as suggested by the CAT assays above. Additionally, the

ratio of virus titers in BHK and HEp-2 cells is shown in the last column. Again, the hrl-

1 virus and the original and reconstructed hrl mutant viruses are clearly deficient for

growth in HEp-2 cells, although hrl-O is not. These results clearly show that both host

range and temperature sensitivity lie in the same mutation, hrl-1 at amino acid 1671 in L.

Table 3-6. Comparative titers of recombinant VSV mutants in BHK and HEp-2 cell
lines at 340C and 400C (Grdzelishvili et al., In Press).
Virus titer Virus titer

(PFU/ml) (PFU/ml) Titer ratio Titer ratio

in BHK cells in HEp-2 cells 340C/400C 340C in

Virus 340C 400C 340C 400C in BHK BHK/HEp-2

Wt 2.6 x109 6.0 x108 6.5 x107 8.0 x106 4.3 40.0

rHR1-0 2.5 x 109 1.0 x 109 4.4 x 107 8.5 x 106 2.5 56.8

rHRI-1 4.2 x 108 < 103 < 10 < 103 > 10s > 10s

rHR1-0,1 1.1 x 109 < 10s < 10s < 10 > 106 > 106

hrl (Orig.) 2.5 x 109 1.5 x 104 < 103 < 10s 1.7 x 105 >106














CHAPTER 4
RESULTS: HR8

Clones

The original hr8 mutant contains five amino acid changes from WT L, ALA in

place of THR at position 148 (hr8-0), ILE in place of THR at position 1097 (hr8-1), ASP

in place of ALA at position 1356 (hr8-2), SER in place of TYR at position 1375 (hr8-3),

and ARG in place of GLY at position 1481 (hr8-4). In order to examine the effect that

each amino acid change has on the function of the L protein, the mutations needed to be

reproduced individually in the L protein. Therefore, each amino acid change was

constructed individually or in combinations and the original hr8 mutant containing all

five mutations was also reconstructed.

Two clones were made prior to the start of this project: hr8-1 and hr8-2. Eight

more clones were successfully produced following the mutagenesis and subcloning

methods described in Chapter 2. They are: hr8-0, hr8-3, hr8-4, hr8-1,2, hr8-1,2,3, hr8-

1,2,3,4, and hr8-0,1,2,3,4. Hr8-0 and hr8-0,1,2,3,4 were later determined to be the

incorrect sequence. Therefore, they as well as hr8-0,1,2,3 were remade by Sherin

Smallwood in our laboratory. All subsequent tests proved that the three new hr8 mutants

were correct (results not shown).

TNT Coupled Transcription/Translation Reaction Analysis of L Proteins

All the clones produced via the Stratagene QuikChange-XL Site-Directed Kit were

tested for full length protein by the TNT system as the amino acid changes introduced

were not meant to truncate the L protein. Those mutants are: hr8-0, hr8-3, hr8-4, hr8-1,2










hr8-1,2,3, hr8-0,1,2,3, hr8-1,2,3,4. Figures 4-1, 4-2, and 4-3 show the products of the

TNTs analyzed by SDS-PAGE. Figure 4-1 indicates that each of the hr8-1,2 and hr8-3

clones are producing full length L protein relative to the WT L control. In Figure 4-2, the

subclones of hr8-1,2 and hr8-1,2,3 are shown and they are also both producing full length

L protein. Figure 4-3 shows a mutant that did not produce full length protein. Hr8-4#2 is

truncated and that mutant was discarded however the other clone is correct. Both hr8-

1,2,3,4 clones produced full length L proteins. Following the TNT reaction, clones that

were producing full length L protein were then sequenced by UF's DNA Sequencing

Core.





o N e

<- L









Figure 4-1. TNT of hr8-1,2 and hr8-3. Analysis of the TNT products by SDS-PAGE
shows that hr8-1,2 and hr8-3 mutants are producing full length L-protein
when compared with WT (control). Numbers 4 and 13 were used in future
experiments.













< L















producing full length L-protein.







<- L










Figure 4-3. TNT of hr8-4 and hr8-1,2,3,4. Analysis of the TNT products by SDS-PAGE
shows that hr8-4 #1 and hr8-1,2,3,4 are producing full length L-protein.

In vivo Expression of L Mutants


All of the L mutants, whether produced via mutagenesis or subcloning must

produce full length L protein in vivo. The in vivo expression system described in Chapter

2 was used to test protein synthesis of the L mutants by radiolabeling infected,

transfected cells. Figure 4-4 compares the L proteins produced by the mutants to the WT

L protein. All the hr8 mutants are producing full length L protein. In other experiments
In ~ ~ *T vioEpeso fLMtnt
All~~~~~~~~~~~~5 ofteLmtnshte rdcdvamtgnsso ucoigms








(data not shown) reconstructed hr8-0, hr8-0,1,2,3 and hr8-0,1,2,3,4 also gave full length
L proteins in cells.



.... CO


Figure 4-4. In vivo expression of hr8 L-mutants. This SDS-PAGE gel shows that the
mutants all produce full length L-protein compared to WT in an in vivo
system. Lanes from left to right: Mock, WT VSV, hr8-0, hr8-1, hr8-2, hr8-3,
hr8-4, hr8-1,2, hr8-1,2,3, hr8-1,2,3,4, hr8-0,1,2,3,4. Hr8-0 and hr8-0,1,2,3,4
were later determined to be incorrect sequences.
Analysis of the hr8 L Mutants for Transcription and Replication
The CAT-ELISA Kit (Roche) was used to test the abilities of the L mutants to
direct replication and transcription from a CAT reporter minigenome compared to WT
VSV L. The CAT minigenome (described in more detail in Chapter 2) is constructed so
that when it is transfected into cells along with N, P and L, chloramphenicol
acetyltransferase protein is produced. The Roche kit measures the amount of translated
CAT produced as a measure of viral RNA synthesis. All CAT assays summarized below
were performed twice on each mutant in triplicate.
Like the original hrl mutant virus, the original hr8 mutant virus showed host
restriction as well as temperature sensitivity. Therefore, CAT assays were performed at
three different temperatures and in three separate cell lines. The permissive and


UUII









nonpermissive temperatures are 340C and 400C, respectively, while 370C represents a

central point between the two where virus is normally grown. BHK cells are permissive,

while the HEp-2 and A549 cell lines are non-permissive.

To test for temperature sensitivity, CAT assays were performed for the L mutants

on HEp-2 cells at all three temperatures. Tables 4-1, 4-2, and 4-3 show the percent of

CAT protein translated by the L mutants relative to that of WT L at 340C, 370C and 400C

respectively. In order to be considered temperature sensitive, the activity of the L

mutants must drop by at least 80% at the higher temperatures. At the permissive

temperature of 340C (Table 4-1), most of the mutants are reduced somewhat but not

below 50% of WT L. Hr8-4 and hr8-0,1,2,3,4 are more reduced to 43% and 34%

respectively. Every mutant shows some effect on RNA synthesis at the permissive

temperature. Surprisingly, as the temperature was increased to 370C and 400C (Tables 4-

2 and 4-3), the mutants apparently become more heat resistant. Especially notable is hr8-

1,2 which is greatly stimulated to 600% of WT L. The reason is unknown but it could be

that WT L is actually less stable at the nonpermissive temperatures than the L mutants

giving higher mutant values. In any case, the results do not point to a specific mutation

as being responsible for the temperature sensitivity of hr8 virus.









Table 4-1. CAT synthesis of hr8 mutants in HEp-2 cells at 340C.


HEp-2 Cells @340C
Mutant Average*
WT 100
hr8-0 61
hr8-1 71
hr8-2 85
hr8-3 50
hr8-4 43
hr8-1,2 ND
hr8-1,2,3 ND
hr8-1,2,3,4 69
hr8-0,1,2,3 52
hr8-0,1,2,3,4 34


*VVT7 infected HEp-2 cells were transfected and incubated at 340C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation. ND Not Done.

Table 4-2. CAT synthesis ofhr8 mutants in HEp-2 cells at 370C.


HEp-2 Cells @370C
Mutant Average*
WT 100
hr8-0 ND
hr8-1 ND
hr8-2 ND
hr8-3 100.5
hr8-4 77.5
hr8-1,2 ND
hr8-1,2,3 79
hr8-1,2,3,4 66
hr8-0,1,2,3 147.5
hr8-0,1,2,3,4 ND


*VVT7 infected HEp-2 cells were transfected and incubated at 370C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation. ND Not Done.











Table 4-3. CAT synthesis of hr8 mutants in HEp-2 cells at 400C.

HEp-2 Cells @400C
Mutant Average*
WT 100
Hr8-0 63
Hr8-1 146
Hr8-2 280
Hr8-3 48.5
Hr8-4 130.5
Hr8-1,2 602.5
Hr8-1,2,3 73
Hr8-1,2,3,4 108.5
Hr8-0,1,2,3 135.5
Hr8-0,1,2,3,4 85

*VVT7 infected HEp-2 cells were transfected and incubated at 400C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation.

To test for host range effects, CAT assays were performed for the L mutants on

A549 and BHK cells at 370C (Tables 4-4 and 4-5). When experiments were conducted

on A549 cells (Table 4-4), the results were largely similar to HEp-2 cells, however hr8-

0,1,2,3 and hr8-0,1,2,3,4 are not included in these results as they were being remade.

Because hr8-4 had somewhat decreased activity in Table 4-2, it was tested along with

hr8-0,1,2,3,4 in the permissive cell line, BHK (Table 4-5). As expected, the mutants

were also more active in the permissive cell line at 370C like in HEp-2 cells at 370C.

Thus the cell line appeared to play no role in the activity of the L protein. Even at a

permissive temperature each of the mutants seemed to have some effect on RNA

synthesis and increased temperatures increased rather than decreased activity. As

indicated in Chapter 3, the vaccinia virus T7 polymerase that infected the cells probably

methylated the mRNAs allowing CAT to be more efficiently translated. Clearly the









minigenome assays with expression of mutant L proteins are not predictive of functions

seen in virus.

Table 4-4. CAT synthesis ofhr8 mutants in A549 cells at 370C.

A549 Cells @370C
Mutant Average*
WT 100
hr8-0 115.5
hr8-1 171.5
hr8-2 107.3
hr8-3 81.5
hr8-4 131
hr8-1,2 153
hr8-1,2,3 74.5
hr8-1,2,3,4 38
hr8-0,1,2,3 ND
hr8-0,1,2,3,4 ND

*VVT7 infected A549 cells were transfected and incubated at 370C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation. ND Not Done

Table 4-5. CAT synthesis ofhr8 mutants in BHK cells at 370C.

BHK Cells @370C
Mutant Average*
WT 100
hr8-4 100
hr8-0,1,2,3,4 41.5

*VVT7 infected BHK cells were transfected and incubated at 370C. Six to nine CAT
assay experiments were averaged and calculated against WT assuming 100% activity
with less than 15% variation.

As with the hrl mutants, Valery Grdzelishvili performed experiments on the hr8 L

mutants that were inserted into recombinant vesicular stomatitis virus. His findings

clearly indicate that both the temperature sensitivity and the host range phenotypes of the

hr8 virus reside in the hr8-4 mutation. The second to last column of Table 4-6 shows that

any recombinant virus containing the -4 mutation is significantly less active at 400C than









at 340C while all the other hr8 mutations are like WT at the nonpermissive temperature.

Additionally, the ratio of titers for each virus in BHK and HEp-2 cells is shown in the last

column. Again, hr8-4, hr8-1,2,3,4, hr8-0,1,2,3,4, and the original hr8 mutant are

deficient in HEp-2 cells relative to BHK cells, while the other mutants are not. These

results clearly show that both host range and temperature sensitivity phenotypes lie in the

same mutation, hr8-4 with a change from GLY to ARG at amino acid 1481.

Table 4-6. Comparative titers of recombinant VSV mutants in BHK and HEp-2 cell
lines at 340C and 400C.

Virus titer Virus titer

(PFU/ml) (PFU/ml) Titer ratio Titer ratio

in BHK cells in HEp-2 cells 340C/400C 340C in

Virus 340C 400C 340C 400C in BHK BHK/HEp-2

Wt 2.6 x109 6.0 x 10 6.5 x107 8.0 x106 4.3 40.0

rHR8-0 8.0 x 10 7.0 x 10 1.5 x 107 1.5 x 106 1.1 53.0

rHR8-1 6.0 x 109 6.0 x 109 1.1x 10 4.5 x 106 1.0 54.5

rHR8-2 2.0 x 10 2.0 x 10 5.5 x 106 1.2 x 106 1.0 36.4

rHR8-3 3.5 x 109 2.0 x 109 1.1 x 10 8.0 x 106 1.8 31.8

rHR8-4 3.0 x 109 < 10 6.0 x 104 < 103 > 3.0 x 106 5.0 x 104

rHR8-0,1,2,3 3.5 x 109 2.0 x 109 3.2 x 10 5.0 x 10 1.7 10.9

rHR8-1,2,3,4 3.2 x 10 < 103 3.0 x 103 < 103 > 105 > 105

rHR8-0,1,2,3,4 2.0 x 109 2.0 x 104 4.0 x 103 < 103 2.0 x 105 5.0 x 105

hr8 (Orig.) 4.0 x 108 < 103 < 103 < 103 > 4.0 x 105 >4.0 x 105














CHAPTER 5
DISCUSSION

The overall focus of my research was to determine which amino acid change or

combination of changes in the hrl and hr8 L mutant viruses were responsible for the host

range and temperature sensitivity phenotypes. The minigenome CAT assays that were

performed on the constructed L mutants which measured the effect of temperature and

host cells on RNA synthesis by the expressed proteins were not completely predictive.

When comparing the CAT assay results to other data obtained in our laboratory, it is clear

that assaying the L protein in cell culture is not the same as assaying the protein in a virus

infection.

The CAT assays suggested that the hrl mutation responsible for temperature

sensitivity is the replacement of VAL for ASP at position 1671 regardless of the cell line

in which it was expressed. The results of Grdzelishvili et al (J. Virology, 2005, in press)

do show that hrl-1 is, in fact, the mutation responsible for temperature sensitivity. His

plaque assay experiments on recombinant VSV, which had the hrl L mutations (Figure

3-6), show that the virus titer ratio at 340C/400C is five logs greater in recombinant hrl-1

and hrl-0,1 and the original hrl mutant indicating that all L proteins containing the hrl-1

mutation are temperature sensitive.

While the CAT assay results were predictive of temperature sensitivity in hrl, it

was unclear which amino acid change was affecting host restriction as all the hrl mutants

had fairly similar activities when compared to WT L regardless of the cell line in which

they were expressed. In nonpermissive cells, such as HEp-2 and A549, the original L









mutant does not methylate mRNA, however, mRNA is methylated in permissive cells,

like BHK (Horikami et al., 1984 and Horikami and Moyer, 1982). This is because viral

transcription occurs in the cytoplasm of the cell. The nucleus of the cell contains

methyltransferase enzymes that are leaked out into the cytoplasm in BHK cells, but not in

nonpermissive cells. Therefore the hrl mutation responsible for unmethylated mRNAs is

rescued by the permissive cells eliminating the appearance of a defect while the non-

permissive cells are unable to rescue methylation and the amount of translated CAT

protein would be decreased. Because the CAT assay requires cells to be infected with

VVT7, which encodes its own methyltransferase enzymes, it is likely that the

recombinant vaccinia virus rescued the methylation in nonpermissive cells eliminating

the appearance of a defect. Thus, this assay cannot measure methylation affects in the L

protein. Grdzelishvili et al. conducted plaque assay experiments to test for host

restriction (J. Virology, 2005, in press) and concluded that hrl-1 is once again the

responsible mutation. The ratios of virus titers in BHK and HEp-2 cells are five logs

greater in hrl-1, hrl-0,1, and the original hrl mutant indicating that L proteins containing

the hrl-1 mutation are host restricted as non-permissive cells are unable to rescue the

methylation defect.

Additional experiments have been conducted by Grdzelishvili to prove that hrl-1 is

in fact causing a methylation defect. He demonstrated in our laboratory that the hrl-1

mutation does not have a significant effect on mRNA synthesis in virus. Additionally,

his results showed that this L mutation did not affect capping of viral mRNA during in

vitro transcription. However, when he conducted in vitro transcription by detergent-

activated purified viruses using a labeled methyl donor (AdoMet), it was clear that hrl-1,









hrl-0,1 and the original hrl mutants were not being methylated at all (results not shown).

Further, when the cap structures of WT and hr 1-0 mutants were analyzed by digesting the

[3H]AdoMet labeled RNA with P1 nuclease to release the cap, the digestion products

were shown to be 7mGpppAm and GpppAm indicating that the non-mutant virus

mRNAs (WT and hrl-0) were being methylated at the cap structure, confirming previous

results with the original hrl virus (Horikami et al., 1984).

The hrl mutation at position 1671 is located in conserved Domain VI of the L

protein. Figure 5-1 indicates the location of Domain VI and also shows the sequence of

several L proteins of other negative stranded RNA viruses with similar conserved

regions. Similar conserved regions in cellular and viral methyltransferases have been

proven to be the S-adenosylmethionine binding site and mutations in this area are known

to abolish the activities of two methylating enzymes, mRNA guanine-N7 cap

methyltransferase and nucleoside-2'-O cap methyltransferase (Luongo et al., 1998, Mao

and Shuman, 1996, Wang and Shuman, 1997). Based on this data, we propose that the

mutation at amino acid 1671 abolishes AdoMet binding and therefore all cap

methylation.








41





SII I iv | v


NNS RNA viruses, L protein
VSV(IN) [gi: 9627234]
VSV(NJ) [gi: 133616]
Rabies [gi:27530026]
Flanders [gi:25140641]
Sendai [gi: 1710716]
Measles [gi: 1041625]
Nipah [gi:13559815]
SV5 [gi: 2981093]
Ebola [gi:33860549]
Marburg [gi: 1350906]


(1650)
(1650)
(1684)
(1666)
(1781)
(1765)
(1820)
(1785)
(1815)
(1931)


.--- hrl: D1671V -
.---

YK-IRSI--LHGMGIHYRDFLS CGDGSGGMTAALLRENVHSRGIFNS
YK-MRTI--ISRLKYPYHDFLACGDGSGGMTAALLRLNRASRGIFNS
YK-LKPI-LDDLNVFPSLCLVVGDGSGGISRAVLNMFPDSKLVFNS
YK-IRSI IENLKITWSYAICGGDGSGGISSYLCRSNPNGKVLFNS
LKALELTYLLSPLVDKDKDRLYLGEGAGAMLSCYDATLGPCINYYNS
YKAVEI STLIRRCLEPGEDGLFLGEGSGSMLITYKEILKLSKCFYNS
YKALNLSPL IQRYL PSGAQRLF IGEGSGSMMLLYQSTLGQSI SFYNS
YKGISCCRYLERLKLPQGDHILYIAEGSGASMTIIEYLFPGRKIYYNS
YKLDEVLWEIESFK-SAVTLAEGEGAGALLL IQK-YQVKTLFFNT
YKLYDLLPPGK-LK-KAICLAEGEGSGARLLLKW--KETDYLFFNT


mRNA guanine-N7 cap MTases


H. sapiens
A. thaliana
D. melanogaster
S. cerevisiae
Vaccinia D1R
Reovirus A2


[gi: 4200033]
[gi:42572501]
[gi:24584376]
[gi: 6319713]
[gi: 126817]
[gi: 127303]


mRNA nucleoside-2'-O cap MTases


Vaccinia VP39
Molluscum poxvirus
West Nile
Yellow fever
Baculovirus
Reovirus A2


[gi: 2554652]
[gi: 903767]
[gi:27735310]
[gi: 9627245]
[gi: 559138]
[gi: 127303]


VLDLGCGKGGDLLKWKKGRI
VLD LACGKGGDLI KWDKARI
VLDMCCGKGGDLLKWEKAAI
VLEILGCGKGGDLRKYGAAGI
VIA IDFGNGADLEKYFYGEI
VLDLGTGPEAKILELIPATC



VVYIGSAPGTHIRYLRDHFY
VLYIGSAPGGHIRYLVEHFR
VVDLGCGRGGWCYYMATQKR
VID LGCGRGGWCYYAAAQKE
FLDLCGGPGEFANYTMSLNP
VAYFGASAGFSGADQPLVIE


Figure 5-1. A) Conserved domains in VSV and the location of the hrl-1 mutation. B)
The same conserved regions in known S-adenosylmethionine binding sites
that are responsible for guanine-N7 cap methyltransferases. C) The same
conserved regions in known S-adenosylmethionine binding sites that are
responsible for nucleoside-2'0 cap methyltransferases.


In the case of hr8, the data on the analysis of viral RNA synthesis were unclear


and unexpected. When testing temperature sensitivity, none of the mutants were


significantly decreased due to an increase in temperature. Surprisingly, hr8-2 and hr8-


1,2 appear to be WT at the permissive temperature but were actually more stable relative


to WT L at 400C. The hr8-2 mutation is found in the nonconserved region between


Domains V and VI at position 1356. Perhaps this mutation changes the overall


conformation of the protein making it more stable. Host restriction results were equally


inconclusive when analyzing the CAT assay results ofhr8. Like hrl, the VVT7 required









for the CAT assays most likely rescued the methylation defect thus eliminating the

appearance of a mutation.

It was impossible to predict which mutant was affected by temperature and the host

cells by reviewing the CAT assay results. However, Grdzelishvili et al. (manuscript in

preparation) have now shown by plaque assays that hr8-4 at position 1488 is the single

mutation responsible for both temperature sensitivity and host restriction. The ratios of

virus titers at 340C/400C and in BHK/HEp-2 are both five log greater for hr8-4, hr8-

1,2,3,4, hr8-0,1,2,3,4 and the original hr8 mutant indicating that any mutant containing

the hr8-4 mutation is both temperature sensitive and host restricted.

Similar experiments that were conducted on hrl are in progress to further test for

the methylation abilities of hr8-4. Grdzelishvili's initial results indicate that hr8-4 is not

temperature sensitive for RNA synthesis but only for methylation. He has found that

methylation is not abolished, as in hrl, but it is decreased by approximately 95%. It was

originally thought that hr8 caused a defect specifically in the 7mG methyltransferase.

Horikami and Moyer (1982) found that hr8 mRNA could be mono-methylated at the

penultimate base or not methylated at all. However, some current results indicate that

hr8 can be methylated at the 7mG position as well as at the 2'0 penultimate base at very

low levels.

The hr8-4 mutation at position 1488 is located in the nonconserved region between

Domains V and VI of the L protein. This change causes a phenotype of limited

methylation. Therefore, position 1488 is not part of the SAM binding site, as partial

methylation can be achieved. It is possible that position 1488 is part of the catalytic site

for one or both of the methylating enzymes. Hr8-4 could also change the overall






43


conformation of the protein thus decreasing its ability to methylate. Another option is

that hr8-4 is part of a catalytic site for one enzyme and changes the overall conformation

of the protein affecting the other methylation event. Grdzelishvili's results, when

completed, will help to determine the function that amino acid 1488 plays in methylation.

Additionally, site-directed mutagenesis will be conducted in the future on the area

between amino acids 1400 and 1671 to further analyze the potential role of the

nonconserved region between Domains V and VI on methylation.














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BIOGRAPHICAL SKETCH


Dallas Lauren Tower was born in Mobile, Alabama, in 1979. In 1990, she moved

to Fairfax, Iowa. After graduating from Cedar Rapids Prairie High School in 1997,

Dallas attended Iowa State University. In December, 2000, she received her Bachelor of

Science degree in biology and started working for Integrated DNA Technologies, Inc.

Dallas moved to Gainesville, Florida in 2002 to pursue graduate education. She received

her Master of Science degree in medical sciences and her Master of Business

Administration degree in May, 2005.