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1 ESCAPE FROM SHORT INTERFERING RNA INDUCED SILENCING IN AN ORTHOBUNYAVIRUS, TENSAW VIRUS By DANIEL MARK FITZPATRICK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUI REMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013
2 2013 Daniel Mark Fitzpatrick
3 To my mother, father, and sister, for all their love and support
4 ACKNOWLEDGMENTS Special thanks g o out to my committee members Dr. Sandra Allan, Dr. James Becnel, and Dr. Rolf Renne. Each of them offered me much appreciated access to their lab, equipment, resources, and the help of their staff and students for various projects. I also wish to thank Dr Michael Scharf, a former committee member, for piquing my interest in the many app lications in ribonucleic acid interference (RNAi) I also want to recognize Dr. Paul Linser, Dr. Lei Zhou, Dr. Drion Boucias, Dr. Monique Coy, and Dr. Doria Bowers for work ing space, cell lines, hands on help, reagents, and all around good cheer. Special thanks go out to Dr. Matthew Avery for all his help with the statistics. I also want to express my deepest gratitude to Dr. James Maruniak and Dr. Alejandra Garcia Maruniak for all they have done. Dr. Maruniak believed in my promise as a budding researcher and an aspiring teacher, and having himself been an excellent example of both, gave me great guidance and encouragement through the ups and downs I have experienced in rese arch and in life in general. Conversely, had it not been for Dr. Garcia Finally, I want to thank my family f or the love and cheerleading that has kept my spirits up along the way. Even though they still make fun of me for studying bugs, I know they are proud of me.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ................................ ..................... 11 Arboviruses ................................ ................................ ................................ ............. 11 Bunyaviridae and Tensaw Virus (TENV) ................................ ................................ 12 Bunyaviridae ................................ ................................ ................................ ..... 12 Tensaw Virus: Prevalence and Distribution ................................ ...................... 1 5 Tensaw Virus Genome ................................ ................................ ..................... 17 Ribonucleic Acid Interference (RNAi) ................................ ................................ ..... 18 Applications of RNA Interference ................................ ................................ ............ 20 Elucidating Gene Function ................................ ................................ ............... 20 Antiviral RNAi ................................ ................................ ................................ ... 22 Evasion of Host Defenses by Ribonucleic Acid (RNA) Viruses ............................... 24 Viral Infection in Alternating Hosts ................................ ................................ .......... 27 2 THE EFFECTS OF ANTIVIRAL RIBONUCLEIC ACID INTERFERENCE ON TENSAW VIRUS REPLICATION ................................ ................................ ............ 33 Intro duction ................................ ................................ ................................ ............. 33 Antiviral R ibonucleic Acid Interference (RNAi) against Arboviruses ................. 33 Fitness of Escape Populations ................................ ................................ ......... 36 Materials and Methods ................................ ................................ ............................ 37 Model Systems ................................ ................................ ................................ 37 Tensaw virus ................................ ................................ .............................. 37 Cell lines ................................ ................................ ................................ .... 38 SiRNA Design and Construction ................................ ................................ ...... 39 Seeding and Transfection of Adherent Cells ................................ .................... 40 Initial Infection with TENV ................................ ................................ ................. 41 Sequencing of Emergent TENV Populations ................................ .................... 41 Determination of Infectious Viral Load ................................ .............................. 41 Confirmation of Sustained RNAi induced Knockdown ................................ ...... 42 Infection with Viral Escape Populations ................................ ............................ 43 Statistical Analysis ................................ ................................ ............................ 43 Results ................................ ................................ ................................ .................... 44 Reduced TENV T iter in HeLa and Vero Cells ................................ ................... 44
6 Sequence Analysis of Emergent TENV in Supernatant ................................ .... 46 Luciferase Assay ................................ ................................ .............................. 47 Infection of Vero, HeLa, and C6/36 Cells with Viral Escape Populations ......... 48 Discussion ................................ ................................ ................................ .............. 49 Initial Escape from Silencing ................................ ................................ ............ 49 Fitness of Escape Populations ................................ ................................ ......... 53 Conclusions ................................ ................................ ................................ ...... 56 APPENDIX A DETERMINING VIRAL TITER BY MEDIAN TISSUE CULTURE INFECTIOUS DOSE (TCID 50 /MILLILITER) ................................ ................................ ................... 69 B LUCIFERASE DEREPRESSION ASSAY ................................ ............................... 72 I. Luciferase Transfection and Expression ................................ .............................. 72 II. Luciferase Quantitation ................................ ................................ ....................... 72 III. Calculation o f Relative Light Units and Example Equation ................................ 73 LIST OF REFERENCES ................................ ................................ ............................... 74 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 87
7 L IST OF TABLES Table page 1 1 Genomic organization of Tensaw (TENV) viral isolate TENV FL06. From Watts et al. (2009) ................................ ................................ .............................. 32 2 1 Growth of Tensaw Passage 1 (TENV P1) isolates from siRNA treated Vero cells ................................ ................................ ................................ .................... 59 2 2 Growth of TENV P1 isolates from siRNA treated HeLa cells ............................. 61 2 3 Growth of TENV from siRNA treated Vero cells in Vero cells ............................. 63 2 4 Growth of TENV from siRNA treated Vero cells in HeLa cells ............................ 65 2 5 Growth of TENV from siRNA treated Vero cells in C6/36 cells ........................... 67
8 LIST OF FIGURES Figure page 1 1 Schematic representati on of the tri partite genome of Tensaw virus (TENV) ...... 31 2 1 Adjusted firefly luciferase ratios ................................ ................................ .......... 58 2 2 Growth of TENV P1 isolates o btained after inf ecting short interfering ( siRNA ) treated Vero cells ................................ ................................ ................................ 60 2 3 Growth of TENV P1 isolates obtained after in fecting siRNA treated HeLa cells ................................ ................................ ................................ .................... 62 2 4 Growth of TENV from siRNA treated Vero cells in Vero cells ............................. 64 2 5 Growth of TENV from siRNA treated Vero cells in HeLa cells ............................ 66 2 6 Growth of TENV from siRNA treated Vero cells in C6/36 cells ........................... 68 A 1 Graphical represen tation of 96 cw plate infected ................................ ................ 71
9 Ab stract 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 ESCAPE FROM SHORT INTERFERING RNA INDUCED SILENCING IN AN ORTHOBUNYAVIRUS, TENSAW VIRUS By Daniel Mark Fitzpatrick May 2013 Chair: James E. Maruniak Major: Entomology and Nematology R ibonucleic acid interference (RNAi) is a major component of antiviral immunity in dipteran insects, including mosquitoes. Virus specific short interfer ing RNA (siRNA) are being considered as a potential therapy to provide resistance to viral infection in vivo However, due to the high mutation rate of ribonucleic acid ( RNA ) viruses, antiviral siRNA treatments often provide incomplete protection against v iral proliferation, which results in a delayed reestablishment of infection. Yet, few studies have examined the infectivity profiles of viral escape populations, especially in arboviruses, where host alternation during arboviral transmission cycles constra ins major genomic shifts. Tensaw virus (Family Bunyaviridae Genus Orthobunyavirus) served as a model system for cell culture experiments examining the effects of inducing the antiviral RNA interference response in vitro on the fitness and sequence of an a rbovirus. Transfection with siRNA targeting the overlapping nucleoprotein/nonstructural protein coding regions of Tensaw virus significantly reduce s viral titer at several time points in Vero (monkey) cells and HeLa (human) cells according to tissue cultu re infective dose assays Virus from various siRNA treated Vero cells were collected at 120 hours post infection (h pi) diluted to uniform viral load, and used to infect both fresh
10 vertebrate (Vero, HeLa) and mosquito (C6/36) cells. Regression analysis of growth curves from all three second passage regimens indicate that the fitness of Tensaw virus from all siRNA treatment conditions that had established infection in Vero cells did not differ significantly from virus that emerged from Tensaw infected Vero cells t hat were not treated with siRNA.
11 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Arboviruses Arthropod borne viruses (arboviruses) are transmitted to hosts through the bite of an infected arthropod. Millions of people worldwide are exposed to arboviruses ea ch year, and research into inner workings of arboviral relationships with their hosts and vectors have been at the forefront of entomological research for decades. Arboviruses are unique insofar as they infect and thrive in both vertebrate and invertebrate hosts, and as such, not only have different pathogeneses and relationships with their respective hosts, but must accordingly be suited to handle the radically different environments of both. Interestingly, the immune responses and the manifestations of in fection differ greatly between vertebrate hosts and invertebrate vectors, which may explain why arboviruses are not typically associated with pathology in the vector (Myles et al. 2008). Pathogenesis in vertebrate hosts ranges from harmless to highly virul ent such as causing hemorrhagic fever or meningoencephalitis, amongst other major illnesses. On the other hand, arboviruses can infect tissue few pathologic effects. Infected vectors tend to have a persistent infection with low levels of virus that can persist for the lifetime of the vector (Brown 1984, Bishop 1996). Accordingly, in cell culture, infection of mammalian cells with arboviruses are often lytic, causing d isruption of cellular replicative functions and cell apoptosis, while in insect cells, infection is typically noncytolytic and persistent (Borucki et al. 2002, Blakqori et al. 2007). It is generally supposed that it is more efficient from an evolutionary s tandpoint for viruses to minimize deleterious effects on vector health rather than to maximize
12 daily survival of the vector and subsequent chances to spread the virus By gaining a better understanding of the molecular biology of arthropod vectors and their interactions with the viruses that inhabit them, scientists can effective arboviral control strategies. Cutting edge molecular approaches are providing considerable information about gene regulation and expression in both viral and host cellular mechanisms. In particular, technologies that involve assessing host immune response and the immune evasion mechanisms of viruses are at the forefront of novel approaches to c ontrolling vector borne viruses. Bunyaviridae and Tensaw Virus (TENV) Bunyaviridae The Bunyaviridae family is a medically important family comprising five genera: Hantavirus Nairovirus Orthobunyavirus Phlebovirus and Tospovirus Of the over 300 describe d bunyaviruses, over 60 species from four genera have been reported to cause disease in humans or livestock, including Crimean Congo hemorrhagic fever (CCHF), Hantavirus (HAN), La Crosse (LAC), and Rift Valley fever (RVF), amongst others (Elliott 1996). O f those five genera, nairoviruses, orthobunyaviruses, and phleboviruses are principally considered arboviruses. Members of the Bunyaviridae family are icosahedral, enveloped viruses with a tripartite negative sense ribonucleic acid ( RNA ) genome comprising small, medium, and large segments, named S, M, and L, respectively (see review in Elliott 1996). The genomes of all five genera encode four essential structural proteins: a nucleoprotein protein, coded for by the N open reading frame (ORF) of the S segment ; two glycoproteins, encoded in the Gn and Gc ORFs on the M segment; and the RNA
13 dependent RNA polymerase (RdRp) encoded by the L ORF on the L segment. Members of the Hantavirus Orthobunyavirus Phlebovirus and Tospovirus genera also contain a nonstructur al protein encoded in the NSs ORF of the S segment (Jskelinen et al. 2007), while genomes of members of the Orthobunyavirus and Tospovirus genera and several members of the Phlebovirus genus, contain a nonstructural protein encoded by the NSm ORF of the M segment (Elliott 1996). Some of the functions of the nonstructural proteins have been elucidated in recent years in reservoir hosts. The protein of the NSm segment has been implicated in inducing cells to create proteinaceous tubular structures that pr omote virion assembly in Bunyamwera virus (BUN) (Genus Orthobunyavirus ) in vertebrate cell lines (Shi et al. 2006, Fontana et al. 2008), and that may facilitate cell to cell movement in tomato spotted wilt virus (TSW) (Genus Tospovirus ) in plants (Storms e t al. 1995). However, the NSm protein was deemed nonessential to viral maturation, replication, and infection in RVF (Genus Phlebovirus ) (Gerrard et al. 2007), and dispensable to growth in Maguari virus (Genus Orthobunyavirus ) (Pollitt et al. 2006). As it stands, though the putative mode of action may vary or may not be fully characterized, the NSs protein serves at least one unambiguous and conserved function in all bunyaviruses studied thus far: attenuation of the reservoir (vertebrate) onses to the virus. Takeda et al. (2002) first demonstrated the immune suppression capabilities of the NSs protein in TSW which attenuates antiviral RNA interference mechanisms of its plant host. Subsequent studies conducted on members of Orthobunyavirus (Blakqori et al. 2007, Hart et al. 2009), Phlebovirus (Billecocq et al. 2004, Perrone et al. 2007, Habjan et al. 2009), and Hantavirus genera
14 (Jskelinen et al. 2007) that infect vertebrate hosts indicate that the NSs protein ymerase II mediated transcription, which in turn blocks the activation of IFN system, the major inducible antiviral response in vertebrates, as well as host cell protein synthesis. Furthermore, Soldan et al. (2005) indicate that expressing the NSs protein in human 293T cells globally suppresses RNA interference induced by short interfering RNA (siRNA). The function of the NSs protein in the mosquito vector is not fully understood. Soldan et al. (2005) demonstrated that siRNA induced gene silencing targeting the overlapping N/NSs region of the S segment significantly attenuated viral expression in Aedes ( Ae.) albopictus C6/36 cells. However, they could not take measures to examine the role of silencing the NSs gene alone because the entire NSs ORF overlaps wi th the N ORF. Blakqori et al. (2007) engineered a recombinant LAC orthobunyavirus that does not express the NSs protein, and they could not detect any advantage that the NSs protein conferred to LAC virus in infecting C6/36 mosquito cells, as the recombina nt LAC did not replicate any less efficiently in C6/36 cells than wild type LAC. Hart et al. (2010) demonstrated similar findings in infection of C6/36 cells, in which NSs deletion mutant of model orthobunyavirus Bunyamwera (BUN) had no effect on mosquito cell transcription or translation. However, more recently, Brackney et al. (2010) and Scott et al. (2010) determined that C6/36 cells mount an incomplete antiviral RNAi response due in part to a deletion in the Dicer 2 gene. Subsequently, Szemiel et al. (2 012) demonstrated that the NSs deletion mutant could replicate effectively in C6/36 cells, no NSs deletion mutant could be detected in RNAi competent Ae. albopictus U4.4 cells, indicating that the BUN NSs gene was essential to establish infection in immune
15 competent cell systems. Furthermore, wild type BUN was able to infect Ae. aegypti salivary glands three days quicker than BUN with the NSs deletion. To date, the specific mechanism by which NSs aids replication of orthobunyaviruses in mosquitoes is unknow n, but NSs does appear to be required for efficacious and productive replication in the vector. Tensaw Virus: Prevalence and Distribution Tensaw virus the model virus to be used in the experiments described herein, is an arbovirus belonging to the Orthobu nyavirus genus of the Bunyaviridae family. TENV was initially isolated from Anopheles (An.) crucians mosquitoes in 1960 in the Tensaw delta valley in Alabama (Sudia et al. 1969). A number of studies in the 1960s, 1970s and 1980s demonstrated the subsequent prevalence and pathogenicity in a wide array of vertebrates. Coleman (1969) detailed animal experiments that exposed several immune competent animals to high doses (10 6. 0 particle forming units per milliliter, or P FU /mL ) of TENV and examined immune respon se. Rabbits and hamsters generated a robust antibody based response to the virus and did not die, while guinea pigs did not produce any demonstrable antibodies to TENV but still survived through the study. Young Swiss mice inoculated with virus at 1 2 days old and 10 days old died with in 3 6 days of infection. Three week old mice died within 6 9 days of infection when TENV was inoculated intracranially, but all survived and produced antibodies to TENV if inoculated intraperi toneally or subcutaneously. Ten w eek old mice inoculated intracranially similarly also survived. Of the hosts studied, only rabbits are found in the wild in the Southeast US.
16 Subsequent tests by Sudia et al. (1969) demonstrated that rabbits, dogs, cats, cotton rats, and hamsters can ampl ify TENV and produce long lasting viremic infections. They then found that experimentally infected dogs produce a high enough viremia that An. quadrimaculatus mosquitoes can take up enough virus to transmit it to immunodeficient suckling mice, indicating t hat dogs can potentially act as reservoir hosts. Interestingly, neither of the birds tested with experimental infection of TENV s parrows or 1 day old chickens produced high viremia, indicating that neither avian host is a particular threat as a reservo ir. Chamberlain et al. (1969), tested blood samples from several wild caught and domestic vertebrates and screened them for antibodies to TENV. They found that 3 of 13 raccoons (23%), 10 of 14 dogs (71%), 1 of 16 cows (6%), and 13 of 150 humans in South Al abama (9%) tested had produced antibodies to TENV, suggesting prior exposure. Subsequent studies by others found that 15% (129 of 850 tested) (Castro et al. 1982), and 22% (65 of 300 tested) (Calisher et al. 1988) of human serum samples from people in Flor ida tested positive for antibodies to TENV in serological tests, suggesting a relatively high endemicity in Florida. Bigler and Hoff (1975) also conducted serological tests of sylvatic mammals, demonstrating that serum from 24% of raccoons (35 of 144 teste d) and two of three marsh rabbits tested positive for TENV antibodies, while no rats mice, opossums, cotton rats, or black rats had antibodies against TENV. They suggested that raccoons and marsh rabbits are often exposed to TENV, but produce a high grade immune response, and suggest that the mosquitoes associated with vectoring TENV do not prefer to feed on mice, opossums, or rats.
17 Calisher et al. (1986) isolated TENV from serum of four different animals, all in Florida dogs cotton rats, swamp rabbits, and cotton mice some of which had not been known to produce antibodies to TENV in previous studies by others. Most importantly, however, virus was isolated from brain tissue of a gray fox in Florida, suggesting that the virus may have produced an enceph alitic infection in the fox. As these produced infective virions in serum and brain tissue samples, all five animals, which are common in Florida, could potentially act as reservoirs for TENV if they produce high enough titers. As for Tensaw related human encephalitis cases, McGowan et al. (1973) reported that of the 3,739 arboviral related encephalitis cases reported from 1955 1971, only one case in Indiana was attributed to TENV infection. Interestingly, however, no other information is provided on the pa tient, and no other arboviral surveillance studies have isolated TENV in Indiana. Tensaw Virus Genome As with other members of the Orthobunyavirus genus, the S segment encodes proteins for a nucleoprotein (N) and a nonstructural protein (NSs) in an overla pping (+1) open reading frame transcribed from the same messenger RNA ( mRNA ) while the M segment encodes two glycoproteins that flank a gene coding a nonstructural protein in non overlapping reading frames (Figure 1 1) (Watts et al. 2009). The putative pr oteins associated with the genomic regions and closest match genome are listed in Table 1 2. TENV was isolated from mosquitoes in Florida for the first time in 2006, and is believed to be distributed throughout the southeast United States (Watts et al. 200 9). Little is known about the function of nonstructural proteins or the structural proteins of the Florida isolate of Tensaw virus. However, it is one of only five completely sequenced
18 viruses in the Orthobunyavirus genus (Watts et al. 2009). Future TENV r esearch will not only provide a better understanding of its biological and molecular characteristics, but additionally, TENV can thus act as a model system for studies into the basic mechanisms of other medically important bunyaviruses. Ribonucleic Acid In terference ( RNAi ) Ribonucleic acid interference ( RNAi ) is a biological process responsible for regulation of endogenous genes as well as detection and destruction of foreign ribonucleic acid ( RNA ) (Fire et al. 1998). Many of the proteins involved in verte brate RNAi pathways have been identified and their roles elucidated. Similarly, analogous proteins with similar functions and sequences have been identified in insects, where the short interfering RNA ( siRNA ) degradation pathway serves as the primary major antiviral defense in insects (Campbell et al. 2008, Cirimotich et al. 2009, Blair 2011), but the proteins have been sequenced for only a few species. RNA viruses generate partially double stranded RNA structures as replicative intermediates in the course of invading and taking over host cellular mechanisms (Westaway et al. 1997). Much like the double stranded RNA (dsRNA) triggered interferon (IFN) pathway in vertebrates, double stranded RNA that is ~30 bp or more in length triggers the RNAi cascade. In in sects, cytoplasmic RNase III enzyme Dicer 2, with the help of dsRNA binding protein R2D2, binds and preferentially cleaves dsRNA from the ends of the strands to generate 21 25 nucleotide (nt) long dsRNA duplexes derived siRNAs, called viRNAs, are then loaded into the multiprotein RNA induced silencing complex (RISC). In the RISC, viRNAs are unwound by an RNA helicase into two single stranded RNA strands. One of the two st rands, deemed the passenger
19 strand, is degraded and discarded, while the remaining strand in the RISC, called the guide strand, that directs the RNA degradation machinery which targets mRNA sequences found throughout the cytoplasm that are complementary to the guide strand. (Paroo et al. 2007, Sanchez Vargas et al. 2004). SiRNA loaded RISCs repress mRNA translation through direct degradation of their mRNA targets (Hamilton et al. 2002). The RNAi signal can then spread between cells, thus inhibiting the repl ication of incoming viruses in neighboring cells by stimulating a preventative antiviral state prior to the dissemination of the virus (Attarzadeh Yazdi et al. 2009). Endogenously expressed small RNAs are responsible for controlling a number of key aspects of gene regulation and cellular development. These small, non coding RNAs are transcribed from the host genome by RNA polymerase II and III from host deoxyribonucleic acid ( DNA ) (Peters and Meister 2007). Complementary sequences then fold back upon themse lves to form double stranded RNA structures, referred to as micro RNAs or miRNAs, which trigger the posttranscriptional downregulation of the expression of protein coding genes within a cell and hence act as internal regulators of gene expression. MicroRNA processing proceeds in a manner similar to siRNA proessing, but with its own set of analogous proteins, including amongst others, separate miRNA processing and cleavage proteins (Dicer 1 and Drosha) and a separate Argonaute protein, AGO1 (Lee et al. 2003, Lee et al. 2004, Okamura et al. 2004). MiRNA loaded RISCs repress the expression of homologous mRNA by direct mRNA degradation and by expediting decay processes that reduce mRNA stability, d Meister 2007, Ghildiyal and Zamore 2009). A few studies have examined the ways that the miRNAs
20 may function in the immune response. Li et al. (2009) identified specific miRNAs expressed in bloodfed Ae. aegypti and suggest that they are involved in immune response. Winter et al. (2007) and Skalsky et al. (2010) also identified miRNAs that are specifically upregulated in response to Plasmodium infection in Anopheles gambiae and West Nile infection in Culex ( Cx .) quinquefasciatus respectively, suggesting th at they may play a role in immune response. However, little is known about the specific function of these miRNAs in mosquito immunity, so further experiments need to be conducted. Applications of RNA Interference Elucidating Gene Function As scientists are gaining a greater understanding of the vast scope of the RNAi host systems in a variety of ways. Systematic analyses of the effects of dsRNA length, secondary struc ture, nucleotide choice and sequence specificity have produced a bevy of information on how to synthesize siRNA duplexes that, when transfected into a variety of in vitro and in vivo systems, effectively trigger the RNAi cascade by mimicking the 21 25 nt D icer cleavage substrate RNA duplex intermediates that are taken up and processed by the RISC (Hammond et al. 2000). The earliest research into RNAi stem from experiments that utilizing dsRNA, and resulted in highly effective silencing of the expression o f endogenous genes identical to the dsRNA introduced (Fire et al. 1998, Bass 2000, Cogoni and Macino 2000). Three 21 25 nt double ngs into in vitro systems, mimicking the siRNA segments created by the Dicer protein, triggers the RISC to knockdown expression of homologous sequences (Elbashir et al. 2001a, b, c). Now, a
21 number of biotechnology companies Dharmacon, Lifetech, Qiagen, and Sigma Genosys, amongst others that synthesize custom siRNA sequences have made these technologies commercially available and relatively affordable to the average researcher. By introducing siRNA to silence specific sequences in a system, many studie s can not only elucidate gene function, but can also examine how these systems change and operate when major components are effectively and precisely shut down. As such, siRNA mediated knockdown of genes has become the hallmark of host gene silencing. Spe cifically, a number of studies have examined introducing siRNA cognate to genes coding for host immune components, which have in turn elucidated many specific aspects of immune function and response as well as many characteristics of pathogenic invasion. H ammond et al. (2001) found that introducing siRNA that depleted the mRNA coding for Argonaute2 (AGO2), the essential Argonaute protein in plant and insect RISC complexes, led to a two to threefold increase in Flock House virus (FHV) RNAs in Nicotiana benth amiana leaves. Similarly, Li et al. (2004) knocked down ago2 expression in Drosophila melanogaster and Anopheles gambiae cell lines, which in turn reestablished infection with recombinant FHV strains that were otherwise uninfective in the cell lines. Exper iments have also been conducted that knocked down immune components in vivo Keene et al. (2004) demonstrated that anopheline mosquitoes injected with dsRNA cognate to AGO 2 nyong virus replication and to viral dissemination Sanchez Vargas et al. (2009) demonstrated that siRNA mediated knockdown of d icer 2 r2d 2 or ago 2 genes that encode essential sensor and effector proteins in the RNAi
22 cascade, increased Dengue virus titers and dec reased the extrinsic incubation period required for transmission of Dengue in A e. aegypti mosquitoes. Antiviral RNAi Mutations add diversity to a viral swarm and can enable a viral population to Theories on viral quasispecies suggest that most RNA viral populations maintain a fairly constant mutation rate, wherein mutations are tolerated at an equilibrium below a deleterious threshold (Domingo et al. 2001). However, Bull et al. (2006) suggest that elevating a ability to remain infective. The concept of lethal mutagenesis hence entails p ushing a viral population within a host beyond that threshold into extinction using mutagens (Bull et al. 2006). Accordingly, many researchers have utilized several technologies, including monoclonal antibodies, ribavirin treatment, and the use of nucleosi de analogues, both in vitro and in vivo with varying success (see Sanjuan et al. 2010 for review). In many instances, the high mutation rates of the viruses allowed for the emergence of viable virus even in the face of treatment (McKeating et al. 1989, Mo et al. 1997, Zharikova et al. 2005, Keck et al. 2009). Since the advent of RNA interference technologies, researchers have explored introducing virus specific dsRNA, siRNA, or DNA constructs or viral vectors coding for short hairpin RNA (shRNA) into cells in order to interfere with the growth and proliferation of viruses. Li et al. (2002) made the landmark observation that viruses trigger RISC in invertebrates to reduce viral gene expression and in turn to halt viral replication. It became apparent through their work and subsequent work by others that
23 RNA interference is a major component of invertebrate antiviral immunity (Campbell et al. 2008, Cirimotich et al. 2009, Blair 2011). Similarly, later studies demonstrated that siRNA can reduce viral gene expr ession in vertebrate systems and that siRNA are too short to trigger an IFN response in some systems (Gitlin et al. 2002, Kapadia et al. 2003). Though the IFN system is a major component of vertebrate antiviral defenses, these studies suggest that RNAi may also play a role in halting viral proliferation. As such, virus specific siRNA are being considered a potential therapy to provide refractoriness in vivo in invertebrates and vertebrates alike (Moens 2009). Subsequently, a number of studies examined the p ossibilities of preventive inoculation with viral specific siRNA as a means for silencing arboviral expression, both in vitro and in vivo in vertebrate and invertebrate systems (Keene et al. 2004, Bai et al. 2005, Murakami et al. 2005, Dash et al. 2008, Wu et al. 2010). Most of these studies revealed that exposure of cell lines to viral specific siRNA prior to infection only transiently suppresses arboviral replication, typically for no more than 72 hours (Bai et Seyhan et al. 2007, Dash et al. 2008). Cell lines are at least transiently resistant to viral infection if they are transfected beforehand with siRNA homologous to regions of a viral genome (Li et al. 2004, Soldan et al. 2005, Wu et al. 2005). However, b ecause RISC binds and/or cleaves viral genomic regions based highly sequence specific binding to the viral RNA target, viruses that were not homologous to the transfected siRNA emerged either through de novo mutations in the targeted sequence (Boden et al. 2003, Das et al. 2004, Soldan et al. 2005) or through the selection for viral clones present in the initial virus population and selected out in the presence of siRNA (Gitlin et al. 2002, Wu et al. 2005). Dash et al.
24 (2008) found that viruses in escape po pulations had the same sequence as the initial wild type virus, but did not examine mechanisms that otherwise allowed for escape from siRNA selection. Wu et al. (2010) also reported a siRNA mediated reduction in cytopathology in Dengue virus (DEN) RNA in C 6/36 Ae. albopictus cells, but RNA levels were decreased by about 40 fold by seven days post infection. In their experiments, antiviral siRNA was transfected into the cells prior to infection, and though the number of viral RNA copies were reduced by singl e dose siRNA transfection, only 8.9% of cells contained antiviral siRNA by seven days after transfection. The authors go on to suggest that siRNA degradation and cell division reduce the concentration of siRNA over time, and thus weakens the antiviral siRN In all of the aforementioned scenarios, viruses were able to circumvent the preventative siRNA treatment and established infection. However, none of these studies included experiments on whether siRNA selection caused any changes to the infectivity of the emergent viral populations. E vasion of Host Defenses by Ribonucleic Acid (RNA) V iruses RNA viruses are particularly prone to genomic drift for a number of reasons. RNA replication is particularly error prone, as there are no know n proofreading or mismatch repair mechanisms in RNA polymerases, and therefore, misinsertion errors arise at a rate of 10 3 to 10 5 substitutions per nucleotide, a much lower fidelity than DNA virus replication (Domingo and Holland 1997). While a vast majo rity of mutations are deleterious (Keightly and Lynch 2003, Sanjuan et al. 2004), this high mutation rate may also confer them with an advantage: an expeditious adaptability by viruses with beneficial mutations to environmental perturbations, particularly those of host immune responses.
25 Several theories have emerged attempting to explain how viruses evade destruction even in the face of robust immune responses. Sawicki et al. (1981) found that within 4 6 hours of infection, Sindbus infected cells were rele asing viable viruses into cell culture supernatant at rates of up to 2000 PFU per cell per hour, which suggests that high replication rates of viruses may overwhelm host immunities by sheer numbers. A bevy of research has also indicated that many arbovirus es and other insect viruses have adapted to the barrage of host immunities by a variety of mechanisms that antagonize vector and host immune components. These mechanisms include proteins that antagonize the vertebrate interferon response (Billecocq et al. 2004, Blakqori et al. 2007, Jskelinen et al. 2007, Perrone et al. 2007, Habjan et al. 2009), or the antiviral RNAi response (Li et al. 2002, Aliyari et al. 2008, Nayak et al. 2010, Qi et al. 2012, van Mierlo et al. 2012), or by encoding subgenomic immun omodulatory RNAs that interfere by competing with the antiviral RNAi response (Schnettler et al. 2012, Schuessler et al. 2012). Furthermore, as mentioned, viruses can also evade the host immune response by de novo mutations that produce viable viral proge ny (Boden et al. 2003, Das et al. 2004, Soldan et al. 2005). Sanjuan et al. (2004) created a series of mutant viruses via site directed mutagenesis of an infectious VSV complementary D NA ( cDNA ) and demonstrated that a majority of single nucleotide substitution mutations, whether randomly chosen or previously described, were lethal or deleterious, but that previously described mutations were more likely to be neutral or beneficial than randomly chosen single nucleotide substitutions. Furth ermore, several studies have indicated that identical point mutations emerge independently in separate lineages of cells infected
26 with identical viruses (Novella et al. 1999, Novella et al. 2004). Taken together, these findings suggest that patterns or mot ifs of mutation arise naturally as a result of parallel evolution, and that these mutations confer an advantage to the mutated clones by helping them evade host defenses. Several studies have ventured to examine changes in the viability of siRNA induced es cape mutants in single host studies with differing results. Senserrich et al. (2007) found that a second passage of human immunodeficiency virus ( HIV 1 ) escape mutants were comparably as fit as their parent strains. Lv et al. (2009), on the other hand, fou nd that FMDV escape mutants were susceptible to siRNA induced degradation when subjected to a second round of selection with the same siRNA Sabariegos et al. (2006 ) found that some HIV 1 escape mutants were more fit, and others less fit, than parent virus depending on which position in the siRNA target region changes. However, examples were not found where virus that escaped siRNA mediated silencing was assayed for fitness in both the vector and the host. Therefore, it has not been thoroughly examined wh ether intracellular immunization with siRNA induces deleterious mutations in the special cases of arboviruses. Since artificially inducing the RNAi pathway with virus specific siRNA can generate an accumulation of mutations and non wild type variations in siRNA targeted regions, it stands to reason that by simulating natural transmission back and forth between hosts, a majority of these escape mutants that may otherwise be able to persist within a single host will not survive when passed to another host.
27 Vi ral Infection in Alternating Hosts Due in large part to low fidelity polymerases, RNA viruses tend to mutate at high rates compared to DNA viruses (Domingo and Holland 1997). However, the consensus sequences of arboviral genomes remain more stable over tim e and express highly variable phenotypes compared to RNA viruses that only infect closely related hosts (Holmes and Twiddy 2003). Though the consensus sequence of a virus may remain the sa me across time, RNA viral population s exhibit a high degree of genot ypic diversity even within a single host. A particular viral variant may not exhibit the highest degree of fitness across all cell and host types, and hence a high degree of genomic diversity may confer fitness to the whole viral population and maintain ph enotypic stability across time (Eigen and Biebricher 1988, van Nimwegen et al. 1999, Wilke et al. 2001). High fitness variants do not always outcompete less fit viral variants during the course of infection within a single host and often may aid in the mai ntenance of minority genomes through complementation, recombination, and reassortment within a cell infected with several variants (Aaskov et al. 2006, Li et al. 2011, Brackney et al. 2011, Ciota et al. 2012). As such, natural selection acts upon the entir e swarm of variants rather than on any particular mutant and maintains genetic diversity within a population accordingly (Eigen and Biebricher 1988, Ciota et al. 2012). Arboviruses are maintained in nature by continuous transmission between vertebrate and arthropod vectors. Whereas a rboviruses tend to maintain a persistent, noncytolytic infection in mosquitoes, which reduces fitness costs associated with infection and allows the vector to transmit virus when feeding, arboviruses cause acute, cytolytic infec tion in mammals, which allows them to produce a high enough titer for transmission to the vector (Scott et al. 1994). Accordingly, these viruses are well suited
28 to very disparate fitness landscapes and must be able to evade highly divergent immune response s. While the phenotype of a virus varies across the range of vectors and hosts it infects, host alternation during the arbovirus life cycle constrains genetic diversity compared to virus that serially infects only one of the two hosts (Greene et al. 2005, Coffey et al. 2008, Vasilakis et al. 2009, Coffey and Vignuzzi 2011). These studies suggest that only a small minority of the clones that emerge in one host will be viable in the other host, and hence by back and forth passage between vertebrate and arthro pod hosts, negative selection acts upon the viral population to provide bottlenecks that continuously purify out the majority of mutants (Domingo and Holland 1997). Experimental studies indicate that this is often the case, as different insertions, deletio ns, or synonymous and nonsynonymous mutations often arise when arboviral populations are subjected to regimens of infection in a vertebrate system only, in an arthropod systems only, or in alternating passage between the two (Novella et al. 1999, Weaver et al. 1999, Greene et al. 2005, Coffey et al. 2008, Vasilakis et al. 2009, Coffey and Vignuzzi 2011, McCurdy et al. 2011, Moutailler et al. 2011). There is a general consensus in the arbovirus literature that an arbovirus infecting a single host cell line i n serial passages will see improved fitness in that cell line passage compared to the parent virus in one step growth curves and competition assays. Weaver et al. (1999), Coffey et al. (2008) and Vasilakis et al. (2009) demonstrate that serial passages of e astern equine encephalitis V enezuelan equine encephalitis and Dengue virus, respectively, in mosquito and/or vertebrate cell lines result in different mutations away from the wild type genomic motif in serial passages, and more consensus mutations in serial passage than seen in alternating passage. Thi s
29 suggests that where serial passage of arboviruses in a single host exhibit different patterns of genetic evolution over time from serial passage though the other host, but that host alternation again limits genomic variance of wild type virus by selectin g for viruses that are most suited to replicating at high rates in both mammalian and mosquito hosts. However, there are several prevailing theories and lines of evidence within the literature about whether there are actual tradeoffs in fitness involved w ith infecting alternating hosts. One school of thought states that generalism by an arbovirus comes at a cost. The low fidelity of the polymerase allows some genetic drift through an accumulation of mutations over time, but host alternation constrains broa d viral population evolution because of antagonistic pleiotropy, where fitness gains exhibited in a serial passage in one host usually results in diminished fitness in the other host (Elena et al. 2009). Accordingly, a virus population will be able to repl icate across several hosts, but will not become optimally fit in any one of those hosts (Elena et al. 2009). The unique and powerful selective pressures of alternating passage of arboviruses on viral variants and of the capabilities of harnessing inducibl e host immunities to knockdown viral expression through RNAi provide us with some novel opportunity to apply experiments that examine the interplay between these bottlenecks. However, there is a paucity of literature on whether conventional antiviral respo nses (e.g., monoclonal antibodies or ribavirin) or novel genetics based treatments like RNAi particularly important if these treatments are utilized as antiviral therapies w ith human patients or in controlling viral infection in vectors. Therefore, the experiments described
30 herein examine whether siRNA based antiviral treatment causes mutations in a model orthobunyavirus, and how this antiviral treatment affects the fitness o f the emergent virus across vertebrate and mosquito cell cultures.
31 Figure 1 1 Schematic representation of the tripartite genome of Tensaw virus (TENV) All three segments are presented in the positive sense. The ex pected proteins coded by each segment, and the amino acid (aa) differences between two TENV isolates [TENV FL06 (first aa) and TENV FE3 66FB (second aa)] as well as the specific aa location are indicated. Checkered region at the beginning of the M coding region, represent the signal peptide. The M segment shows the translated polyprotein before (solid bar) and after cleavage (open boxes). Lines flanking the boxed areas represent the the general location of the four conserved regions found in RdRp. Figure from Watts et al. (2009).
32 Table 1 1 Genomic organization of Tensaw (TENV) viral isolate TENV FL06 From Watts et al. (2009) Segment ( nucleotide size) Nucleotide Po sition of mRNA Size in nucleotides Size in amino acids Protein encoded Small (946) 75 776 702 233 Nucleocapsid 94 399 306 101 Nonstructural protein (NSs) 481 696 216 71 Uncharacterized ORF Medium (4461) 44 4354 4311 1436 Polyprotein precu rsor 17 Signal peptide 286 Glycoprotein (G n ) 175 Nonstructural protein (NSm) 958 Glycoprotein (G c ) Large (6864) 50 6766 6716 2238 RNA dependent RNA polymerase
33 CHAPTER 2 THE EFFECTS OF ANTIVIRAL RIBONUCLEIC ACID INTERFERENCE ON TENSAW VIR US REPLICATION Introduction Antiviral R i bonucleic Acid Interference (RNAi) a gainst A rboviruses Viruses from all three major families of mosquito transmitted arboviruses Togaviridae Flaviviridae and Bunyaviridae are responsive to antiviral ribonucleic acid interfe rence (RNAi). In general, these studies indicate that RNAi efficacy varies tremendously, based upon factors like viral target choice, the method of RNAi induction (i.e., whether inverted repeat ribonucleic acid [ RNA ] short interfering RNA [ siRNA ] or shor t hairpin RNA [ shRNA ] expressing plasmids are used), and model system. A major complication associated with this is escape from silencing, in which virus continues to proliferate in the face of antiviral RNAi therapy. Because of the mutability of RNA virus es, they can utilize a number of mechanisms to evade the RNAi response, including induction of or selection for mutations, insertions and deletions at the target site or in viral promoter and enhancer regions that abrogate the sequence specific interferenc e (see Shah and Schaffer 2011 for review). However, genomic changes are not responsible for all cases of viral escape (Gitlin et al. 2002, Soldan et al. 2005, Wu et al. 2005, Dash et al. 2008). A majority of the antiviral RNAi literature pertaining to ar boviruses focus on viruses in the Togaviridae and Flaviviridae families, and as such, there are only a few studies that investigate the therapeutic capacities of RNAi on medically important Bunyaviridae In these studies, both structural and non structural components of the Bunyaviridae have been targeted with varying degrees of success.
34 Levin et al. (2006) transfected three siRNA specific to Akabane orthobunyavirus S segment 24 hours prior to infection (hpi) and observed 99% reduction in viral proliferation ac cording to quantitative reverse transcriptase polymerase chain reaction ( RT PCR ) immunofluorescence, and endpoint virus titration up to 48 hpi. Soldan et al. (2005) pretreated human 293T cells and mosquito C6/36 cells with single siRNAs specific to all three segments of La Crosse orthobunyavirus (LAC), a nd reported a reduction in plaque forming units (PFU) between 0 2.5 log knockdown for 12 of 13 siRNAs tested, but found up to a 3.8 log reduction in plaque forming units ( PFU ) at 72 hpi in cells that were pre inoculated with siRNA specific to the overlapping NSs/N reading frame. Interestingly, the authors found a considerably higher sequence diversity at the siRNA target region at 72 hpi in LAC targeted with S specific and M specific siRNA, and observed viral variants in siRNA treated samples that were not found in mock treated i nfections. This suggests that minority variants not seen in untreated cells were selected for in response to targeting the wild type, or the de novo induction of variants not present in the viral population prior to treatment. Flusin et al. (2012) also tar geted all three segments with antiviral siRNA and found that S specific siRNA most efficiently inhibited Hazara nairovirus nucleoprotein production up to 48 hpi, according to e nzyme linked immuno sorbent assay ( ELISA ) and Western blot assays. Scott et al. (2012) designed plasmids coding for s hort hairpin RNA (shRNA) specific to the non overlapping NSs and N genes of Rift Valley Fever virus (RVF), in the genus Phlebovirus Interestingly, the shRNA cassettes were able to significantly reduce the expression of plasmids coding for the NSs and N pr oteins and to abrogate the cytotoxic effects of the NSs protein. However, shRNA induced therapeutic
35 prevention of RVF infection in cell culture was minimal. According to ELISA assays, N specific shRNA only marginally reduced the expression of the putative nucleoprotein (15.5% by 72 hpi), whereas NSs specific shRNA did not affect the production of nucleoprotein protein by RVF infected cells. These results suggest that while shRNA expressing plasmids may appear to eff ectively target bunyaviral RNA sequences f or destruction, RNAi may not necessarily inhibit viral proliferation during active infection, where virus may eventually overcome antiviral treatment. Because of the paucity of literature on how bunyaviruses respond to antiviral RNAi, questions about its e fficacy remain unanswered. Most of these studies treated knockdown as a binary event; either knockdown was achieved in the first 48 72 hours following initial infection or it was not. Little is known about RNAi induced knockdown of bunyaviruses at time poi nts after 72 hours. Soldan et al. (2005) saw an increase in LAC titer beginning between 48 and 72 hours in many siRNAs tested, suggesting the reestablishment of infection after initially successful knockdown; however, none of their preventative siRNA assay s provided data beyond 72 hpi. Similarly, in Levin et al. (2006), virus replication was reduced by siRNA pre treatment but still apparent between 24 and 48 hpi according to RT PCR and viral titration assays, but there was no information on the fate of viru s replication thereafter. Because few studies have examined the effects of antiviral siRNA on bunyaviruses beyond 72 hours, it is not known whether these siRNA halt infection altogether or whether treatment simply delays the onset of infection, or whether emergent virus is perhaps immune to the targeting siRNA. Furthermore, of these studies, only Soldan et al. (2005) examined the effect of siRNA treatment on the sequence of the virus.
36 Fitness of Escape P opulations Antiviral RNAi has been shown to affect the viability of emergent virus in several model systems, and those fitness differences depended primarily on whether RNAi selection was maintained following escape from silencing. Gitlin et al. (2005) utilized viral competition assays to demonstrate that pol iovirus that escaped siRNA silencing in HeLa cells was only more fit than wild type poliovirus when siRNA selection remained in place; otherwise, RNAi resistant variants were no more fit than wild type virus in untreated cells. Beyond the Gitlin et al (200 5) study, most of the work on the viability of escape mutants has been performed in HIV research, where there is a history of utilizing siRNA and shRNA induced silencing to reliably select for or induce mutations that have reduced fitness. Some escape muta nts in HIV 1 have been well characterized and can be reliably induced. Accordingly second generation shRNA have been designed to anticipate and suppress potential escape mutants (Sabariegos et al. 2006 Schopman et al. 2010, Shah et al. 2012, Schopman et al. 2012 ) Similarly, after identifying common escape variants in poliovirus, Gitlin et al. (2005) designed pooled siRNA that included siRNA against those variants, which resulted in efficacious suppression of viral replication. Hence, there are precedents fo r using patterns by which a virus escapes RNAi induced silencing to create other effective treatments. However, where antiviral RNAi in HIV 1 has been studied thoroughly, no research could be found about the fitness of escape populations in the arboviral f amilies. Furthermore, antiviral RNAi studies have focused largely on one host systems (e.g., HIV 1, poliovirus), where escape from silencing has focused on the viability of emergent virus in the same cells Apparently no studies have examined the effects of
37 antiviral RNAi on fitness across a two or more host system that requires host alternation to maintain transmission. Indeed, the stabilizing selection of alternating passage has been demonstrated in vitro and in vivo (Greene et al. 2005, Coffey et al. 20 08, Vasilakis et al. 2009, Coffey and Vignuzzi 2011). The research in this chapter examines how preventative antiviral siRNA treatment affects the siRNA targeted region and the infectivity of Tensaw virus (TENV) an orthobunyavirus, for up to 120 hpi in s everal cell culture model systems. These findings will provide insight into how bunyaviruses are affected by antiviral RNAi therapy over a longer period of infection that described in previous studies. Moreover, this study analyzes how antiviral RNAi affec ts the fitness of TENV emerging from siRNA treated monkey kidney cells (Vero) in three model systems: two vertebrate (Vero, human hepatocellular carcinoma HeLa cells) and one mosquito ( Ae albopictus C6/36) cell lines. Materials and Methods Model Systems Tensaw virus Tensaw virus isolate TENV FL06 was isolated from mosquitoes collected in Alachua County, Florida in 2006 as described in Watts et al. (2009). The initial stock was generated when mosquito homogenate was inoculated in 1 X 10 5 Vero cells growin 15 media containing 5% fetal bovine serum (FBS), gentamycin, penicillin streptomycin sulfate solution, and amphotericin B. Following visible cytopathology at 6 days post infection ( dpi ) viral supernatant was used to infect 7 x 10 5 Vero cells in T 25 flasks. Twenty four hours later, viral supernatants were collected and frozen at 7 0 C Virus titer was then determined with endpoint dilution titration to
38 determine mean tissue culture infectious dose per milliliter (TCID 50 /mL) (Appendix A) Tensaw virus stocks generated by this method contained approximately 1 X 10 7 TCID 50 /mL. Cell l ines assessing the effects of viral replication in an in vitro model. Three cell line s will act as models for host systems in my studies. HeLa cells, a human cancer cell line isolated from Henrietta Lacks in 1951, have stood in as a popular in vitro model for human cancer and virology studies for more than 50 years (Lucey et al. 2009). Ca ntell (1961) demonstrated that HeLa cells display a robust IFN response to viral infection, and as such, our lab has utilized these cells in a number of experiments to assess the effects of viruses in immune competent vertebrate cell lines. HeLa cells were maintained at 37 C incubators without carbon dioxide ( CO 2 ) 15 media with 5% fetal bovine serum (FBS) and 50 micrograms per milliliter ( g/mL ) gentamycin unless otherwise stated. Vero cells are a continuous aneuploid derived from the epithelial kidney cells of African green monkey, Cercopithecus aethiops Vero cells cannot produce IFN because of a genetic deletion, and as such have an attenuated interferon based antiviral response (Desmyter et al. 1968, Emeny and Morgan 1979, Chew et al. 2009). Vero cells are used by our lab because they are highly perm issive to a range of viruses and because of their observable cytopathologic effects (CPE) caused by infection. Vero cells were maintained at 37 C incubators without CO 2 15 media with 5% FBS and 50 g/mL gentamycin unless otherwis e stated.
39 C6/36 cells, derived from larval Ae. albopictus mosquitoes by Singh (1967), are used in many studies as models for viral infection, as they are susceptible to infection by a wide range of viruses (Chen et al. 2004a, Cirimotich et al. 2009). Brack ney et al. (2010) demonstrated that C6/36 cells have an incomplete RNA interference immune response to viruses. Scott et al. (2010) have identified a single deletion at nucleotide (nt) 2460 in the complementary deoxyribonucleic acid (cDNA) of the C6/36 Dic er 2 gene a major component of the antiviral RNAi response causing a frame shift and accordingly an early termination codon. This paper demonstrates that the putative Dicer 2 protein is dysfunctional, suggesting that this dysfunction may contribute to C 6/36 cells producing high titers of virus (Brackney et al. 2010, Scott et al. 2010). However, ~22 nt anti GFP siRNA is introduced, there is a reduc tion in GFP expression in C6/36 cells suggesting that RNAi machinery downstream of the Dicer complex is func tional, and that transfected siRNA can still knockdown gene expression (Brackney et al. 2010). C6/36 cells were maintained in 26 C incubators without CO 2 buffering in 15 media with 5% FBS and 50 g/mL gentamycin unless otherwise stated. SiRNA Design and Construction Candidate virus specific siRNA were provided according to criteria laid out by Tuschl et al. (2004) by Sigma Aldrich using algorithms that take into account secondary structure, G C content, and particular purine pyramidine prefere nces at specific positions in the siRNA. SiRNA s were chosen for use in our work from candidate siRNA sequences provided by Sigma Aldrich that we further analyzed with Mfold, a web based program that predicts secondary folding of target mRNAs according to t hermodynamics (Zuker et al. 2009), and according to genomic sequence conservation across viruses in the Bunyaviridae family (Elliott 1996), in order that we may specifically target less
40 mutable viral sequences that are required for the function of putative viral proteins. The two Tens aw spe cific siRNA sequences are: NSs siRNA : targeted region (cDNA): 5' GTCTTACCCTCCACAGGCT sense siRNA: 5' GUCUUACCCUCCACAGGCUdTdT antisense siRNA: 5' AGCCUGUGGAGGGUAAGACdTdT RdRp siRNA : targeted region (cDNA): 5' AGCTTGAAATAAATGCAGA sense siRNA: 5' AGCUUGAAAUAAAUGCAGAdTdT antisense siRNA: 5' UCUGCAUUUAUUUCAAGCUdTdT NSs siRNA is homologous to nt 340 358 on the S segment and lies on a conserved region of the overlapping N/NSs open reading frames accordi ng to alignment with other viruses in the Bunyamwera serogroup. Similarly, RdRp siRNA is homologous to nt 3141 3159, and aligns with one of four polymerase motifs that are also conserved amongst orthobunyaviruses (Poch et al. 1989). A negative control (no nsense) siRNA was purchased from Lifetech, and was analyzed by Lifetech technical support to ensure that the siRNA had no homology to sequences in the Tensaw viral genome or the genomes of any cell lines used in our studies. Seeding and Transfection of Adh erent Cells Vero, HeLa, or C6/36 cells were seeded in 24 cluster well plates at 6 X 10 4 cells per well approximately 24 hours prior to transfection. TENV specific or nonsense siRNA s were transfected into Vero or HeLa cells with the lipid based cationic lip osome ( Mirus Bio) per manufacturer instructions, approximately 24 h ours prior to infection with TENV
41 Initial I nfection with TENV Cells were infected with 150 L of media containing T ENV at multiplicity of infection of 0.01 ( MOI 0.01 ) in Vero cells or at MOI 0.1 in HeLa cells for 1 h our Viral supernatant was then removed from the cells, and the cells were washed twice with 1X media was then added to cells and then incubated at 37 C C ell supernatants and whole cell lysates were collected separately at 0, 24, 48, 72, 96, and 120 hpi, and stored at 70 C until subsequent assays were performed. Sequencing of Emergent TENV P opulations RNA was extracted with TRI zol reagent from supernatants collected at 24, 48, 72, 96, and 120 hpi from TENV infected Vero and HeLa cells that had been transfected with TENV NSs siRNA prior to infection. RT PCR was performed on extracted RNA with primers that amplified the entire NSs gene, which contains the NSs siRNA target region. The resultant amplicon was cloned into the pGEM T Easy Vector (Promega) and transformed into Max E. coli cells (Lifetech), which were grown overnight on selective media. Colonies containing the cloned plasmid were g rown in selective media, and the siRNA target region of the plasmid was sequenced at the UF Interdisciplinary Center for Biotechnology Research (ICBR) using BigDye Terminator Chemistry v1.0 (ABI). Determination of Infectious Viral Load Infectious viral l oad was determined by endpoint dilution titration to determine the mean tissue culture infectious dose per m L (TCID 50 /mL) in viral supernatants (see Appendix A for protocol and calculation of titer).
42 Confirmation of Sustained RNAi induced K nockdown The Dua l Luciferase Reporter Assay System (Promega) allows for the quantitation of the expression of two exogenous luminescent signals, firefly luciferase and Renilla luciferase, from cellular lysates. Both luciferase enzymes are active without post translationa l modification, retain enzymatic activity in the cellular lysate, and produce a luminescent signal when exposed to their respective substrates. This signal can be quantified by a microplate reader. This assay was modified and optimized in Skalsky et al. (2 007) and Haecker et al. (2012) to examine knockdown efficiency in mammalian cell cultures. To do this, cells are cotransfected with firefly and Renilla luciferases. The modification and validation of the pGL3 vector containing the firefly luciferase is des cribed in full in Skalsky et al. (2007). In short, nucleotides 658 to 2495 1 gene containing four miRNA binding sites for the K12 11 microRNA mimic (miR K12 11) was cloned downstream of the firefly luciferase gene coding regi on. Therefore, miR K12 11, which utilizes the cellular RNA interference machinery similar to the antiviral siRNA in this study suppresses the expression of firefly luciferase. Accordingly, miR K12 11 is co transfected with the two luciferases in the treat ment condition. At the appropriate time point following transfection, cells are lysed with a lysis buffer, and the ratio of the expression of the firefly and Renilla luciferases are compared in the presence and in the absence of the microRNA to quantitate the relative reduction of the firefly luciferase expression. Transfection of Vero cells for the luciferase knockdown assay is described in full in Appendix B. Because the transfection infection protocols examined virus knockdown at 132 hours after the ini tial transfection i.e., 24 hours for transfection, followed by 120 hours of infection cells in the luciferase knockdown assay were harvested accordingly
43 with Passive Lysis Buffer (Promega) and the activity of the two luciferases were quantified in suc cession using the Dual Luciferase Reporter Assay kit (Promega) Dr. using an MLX Microplate Luminometer (Dynex). Infection with Viral Escape Populations In order to assess the infectivity of virus emerging from siRNA treated cells across several hosts Vero, HeLa, or C6/36 cells were seeded in 24 cluster well plates at 8 X 10 4 cells per well approximately 24 h ours prior to infection. Once the TCID 50 /mL was determined for cell supernatants from each of the four Vero cell treatments from Passage 1 (NSs siRNA, RdRp siRNA, nonsense siRNA, and no treatment with siRNA or transfection reagent), stocks stored at 70 C were then thawed and diluted accordingly with L 0.01 or untr eate d HeLa or C6/36 cells at MOI 0. 1 for 1 h our with emergent virus from all four Vero P1 treatments using methods described above for initial infection with TENV. Cell supernatants for all second passage infections were collected every 24 h ours up to 120 hpi, an d stored at 70 C until endpoint dilution assays (TCID 50 /mL) to assess titer were performed as described in Appendix A. Three or four replicates of infection of Vero, HeLa, C6/36 with Vero P1 escape populations were performed. Statistic al Analysis A mixed effects model was used for analysis of all viral g rowth data using the lme4 package and the lmer function in the R software environment, version 2.15.2, with time and siRNA treatment condition predictor variables. Time was treated as continuous, and linea r and quadratic terms were included in the model. Interaction effects for siRNA treatment condition with both linear and quadratic time trends were also included. Random effects for replicate and treatment batch are included to account
44 for random variabili ty due to these factors. For the analysis of the growth curve of Vero escape populations in C6/36 cells, a likelihood ratio test comparing a linear only model with the quadratic model failed to reject the null hypothesis that d ata fit both models equally w ell, so the simpler (i.e., linear) model was used. For all other growth curves, a quadratic model was used for analysi s. To account for multiple comparisons, the method described by Benjamini and Hochberg (1995) was applied to the pairwise differences anal yses to adjust for false discovery rate. Therefore, the values have been adjusted accordingly, so that a p value of 0.05 or lower is still considered significant for all growth curve analysis data. test calculated with Microsoft Excel was use d to analyze the luciferase repression assay data, and a p value of 0.05 or lower was considered significant. Results Reduced TENV Titer in HeLa and Vero C ells To assess how TENV viral replication is affected by transfection with TENV specific siRNA, Vero and HeLa cells previously transfected with NSs siRNA or RdRp siRNA or with nonsense siRNA were infected with TENV at MOI 0.01 and MOI 0.1, respectively R esults from the primary TENV infection (referred to as Passage 1 or P1) are shown in Figure 2 2 and 2 3 In Vero cells infected with TENV, there w ere no significant difference s in viral titer s in cells previously transfected with nonsense siRNA and untransfected cells (p > 0.082) at any time point which indicate d that the transfection reagent and nonsense siRNA d id not affect viral proliferation in Vero cells. Transfection of Vero cells with NSs siRNA resulted in a reduction in TENV titer s that w ere significantly lower than the no siRNA condition at all time points (p < 0.002) and
45 significantly lower than nonsense siRNA time points at all time points except 96 hpi, in which there was a borderline significance (p < 0.048 for 24, 48, 72, and 120 hpi; p = 0.082 for 96 hpi). However, though significantly lower than the untreated control at all time points, vira l titer in NSs siRNA treated cells did increase at each point after t = 24 hpi, and in fact showed an increase in TENV titer of nearly four logs between 48 and 120 hpi. This increase was the highest increase seen across conditions in that span of time. How ever, r egression analysis of the time trends of the four growth curves from 0 120 hpi did not indicate that there were significant differences between any treatments in the rate of TENV growth over time (p > 0.908) Whereas TENV titer was lower as a result of NSs siRNA treatment throughout the 120 hours of exposure TENV titer in RdRp siRNA treated Vero cells was significantly lower than the infection only cells at all points 48 hpi and later (p < 0.040), but was never significantly different from the nonse nse siRNA treated Vero cells at any time point (p > 0.154), suggesting that there is no additive silencing effect induced by RdRp siRNA in Vero cells. HeLa cells were transfected with the same three siRNA (NSs siRNA, RdRp siRNA or nonsense siRNA) prior to infection with TENV FL06, and the resulting TENV P1 supernatants, collected from 0 to 120 hpi then titered in Vero cells. TENV infected HeLa cells that were not treated with transfection reagent or siRNA had a significantly higher titer than cells treated with NSs siRNA, RdRp siRNA or nonsense siRNA at all time points (p < 0.033) (Figure 2 3). This indicate d that there was a significant effect of treatment with siRNA and transfection reagent, because i t was higher at all time points for all conditions than even the nonsense siRNA control Transfection of HeLa cells with
46 RdRp siRNA did not result in a significant reduction in viral titer compared to viral infection in nonsense siRNA treated HeLa cells at any time point, suggesting again that RdRp siRNA does n ot confer any significant antiviral resistance. On the other hand, NSs siRNA treatment resulted in viral titer significantly lower than that of infected cells without siRNA treatment at all time points (p < 0.0002) and lower than nonsense siRNA treated cel ls up to 96 hpi (p < 0.003 up to 96 hpi; p = 0.11 at 120 hpi). In fact, TENV titer increased only 1.5 log during the 120 h ours of infection when treated with NSs siRNA, compared to a 2.3 or more log increase in all other RdRp siRNA treatments. Finally, regress ion analysis indicate d that the rate of TENV growth differed between all conditions (p < 0.039), aside from the RdRp siRNA and nonsense siRNA growth curves, which were not significantly different (p = 0.861). In other words, where transfection with siRNA n onspecifically reduces the rate of TENV growth in HeLa cells compared to infection in untransfected HeLa, NSs siRNA reduces the rate of TENV growth even more. These results show ed that NSs siRNA wa s capable of causing a decrease in viral titer in both vert ebrate cell lines across time, though NSs siRNA only significantly reduced the rate of growth in HeLa cells. Sequence A nalysis of E mergent TENV in S upernatant To determine if the increase in the number of infectious virions that followed at 72 h ours after NSs siRNA treatment in Vero cells was due to the selection for non wild type variants the NSs gene was sequenced and compared to the TENV P1 virus that was collected from siRNA untreated cells (TENV P1V). The results from a small number of clones (n = 9, 10 27, in the three replicates tested) showed that only one TENV isolate from NSs siRNA treated cells had sequence modifications that alter the siRNA target
47 sequence. One had a point mutation (from GTCTTACCCTCCACAGGCT to GTCT C ACCCTCCACAGGCT) that would resu lt in no amino acid replacement for the NSs gene, but would cause a replacement in the nucleoprotein (N) gene from a leucine to a proline. All the viral clones belonging to the untreated controls tested (n=10) showed no nucleotide modification in the NSs s iRNA target region. The altered amino acid presumably should not affect the function of the TENV nucleoprotein, since other orthobunyaviruses have been sequenced containing a proline in the same location of the translated protein (Yandoko et al. 2007). Mor eover, in all three instances where TENV NSs was sequenced, there were no consensus mutations in the regions flanking the NSs siRNA target site that would indicate changes to viral RNA secondary structure. Subsequent analysis of seven to ten sequences of T ENV emerging from TENV NSs siRNA in infected HeLa or Vero cells at 24, 48, 72, 96, and 120 hpi did not reveal any changes to the target region of siRNA treated TENV at any time point, suggesting that changes to the siRNA target sequence were not always ass ociated with escape from silencing with antiviral siRNA in this system. Luciferase Assay In order to confirm that RNAi induced expression persists in Vero cells for the length of the experiment, a dual luciferase repression assays were performed in which p lasmids coding for firefly luciferase and Renilla luciferase we re co transfected with a microRNA mimic (miR K12 11) that suppresses expression of firefly luciferase. At 132 hours after transfection, firefly luciferase expression was reduced 96.35% compared to cells that were transfected with the luciferase reporter plasmids only ( See Figure 2 1 ). However, because of the high variation in the no microRNA condition, the differences between conditions were only borderline significant (p = 0.052).
48 Infection of Vero, HeLa, and C6/36 Cells with Viral Escape Populations To assess whether antiviral siRNA treatment affects replication in several cell culture systems, TENV supernatants collected from the four treatments described NSs siRNA, RdRp siRNA, nonsense siR NA, and no siRNA treatments at 120 hpi were diluted to uniform viral loads, and used to infect Vero, HeLa, or C6/36 cells. Growth curves and TCID 50 /mL data for all three second passage infection regimens are shown in Figures 2 4, 2 5, and 2 6. All the i nformation concerning the TSV P1 virus that had been used as inoculum is presented in parenthesis including the specific siRNA used to transfect cells prior to the first infection and the time of the TSV represents TENV Passage 2 (P2) virus in HeLa cells (H) that were infected with a P1 inoculum collected from infected Vero cells (P1V) at 120 hpi after those cells had been transfected with the NSs siRNA. In all three passage regimens, no significant diffe rences were apparent in growth rate of P1 vi rus from either TENV specific si RNA treatments compared to virus from untreated or nonsense siRNA treated Vero cells, according to regression analysis of viral growth over time. (p > 0.717 for Vero to Vero passag e; p > 0.103 for Vero to C6/36 passage; p > 0.596 for Vero to HeLa passage). Moreover, there were no significant differences in viral load between the four treatments at any time points in Vero to C6/36 passage (p > 0.538) or Vero to HeLa passage (p > 0.19 9). In the Vero to Vero passage, the titer of TENV from RdRP siRNA treated cells was lower than TENV from untreated cells at 24 hpi (p = 0.035), but this difference disappeared in all subsequent time points. Otherwise, no other pairwise comparisons achieve d statistical significance in Vero to Vero passage regimen (p > 0.057).
49 Discussion Initial Escape from S ilencing To date no studies have examined whether siRNA escape populations retained infectivity across hosts, and so our study examined how transfecti on of two siRNA homologous to conserved regions of TENV (NSs siRNA and RdRp siRNA) into Vero monkey cells affected viral growth in vertebrate and mosquito cell cultures, using change in viral titer in fresh cell cultures over time as a metric for fitness. RdRp siRNA did not inhibit viral replication any more than control siRNA in either Vero or HeLa cells. This is interesting for several reasons. It has been demonstrated that the L mRNA exists in the lowest quantity in other orthobunyavirus infected cells ( Rossier et al. 1988), and that the L segment product (the viral polymerase) is necessary for replication and transcription of the rest of the segments. Therefore it was believed that interfering with the typically low expression of the L segment would effi ciently reduce general virus expression. Furthermore, sequence analysis determined that the RdRp siRNA target region was highly conserved among orthobunyaviruses, While the function of this domain remains unclear in orthobunyaviruses conservation of seque nce would suggest that it is a functional domain, and therefore would be expected to be resistant to mutations that would otherwise allow for loss of sequence specific interference. That said, other RNAi studies targeted other conserved domains on the buny aviral L segment, and therefore there is no other precedent for antiviral RNAi against the putative functional domain chosen in this study. Where the L segment was targeted other studies, the efficiency of antiviral RNAi induced knockdown varied widely, de pending on the target chosen, even in highly conserved viral coding regions. For instance, d epending on which region of the L segment they targeted with siRNA Soldan
50 et al. (2005) observed between two fold and forty fold reduction in La Crosse titer in 29 3T and C6/36 cells compared to control siRNA treated cells at 48 hours. Similarly, Flusin et al. (2012) saw significant knockdown of Akabane infection with only one of four L specific siRNAs tested in human A549 cells Several explanations are possible. Bu nyaviruses express nucleoproteins that form ribonucleoprotein complexes with the genomic and antigenomic RNA (Elliott 1996). This may have prevented access of the RNA interference silencing complex ( RISC ) machinery to naked viral RNA. Additionally, because the guide strand of the RISC complex must bind to viral RNA to ac tivate translational repression or mRNA degradation, secondary structures in or near the target region viral RNA may have similarly prevented adequate binding of the RISC complex to the target site. On the other hand, siRNA aga inst the S segment of Tensaw was effective at reducing viral titer in both Vero and HeLa cells. These findings were consistent with the findings of others in S specific antiviral RNAi against bunyaviruses (Soldan et al. 2005, Levin et al. 2006, Flusin et a l. 2012). Other studies have demonstrated that transfection in HeLa cells induces a non specific antiviral interferon response to transfection reagents and siRNA (Li et al. 1998, Reynold s et al. 2006). Therefore, because RdRp siRNA treatment did not reduce viral titer any more than nonsense siRNA treatment, this suggests that the RdRp siRNA related reduction in TENV titer wa s not a result of increased virus specific knockdown of viral gene expression by the RISC, but the result of general induction of the i nterferon response. NSs siRNA reduced TENV growth even further than the nonspecific siRNA response, suggesting that NSs siRNA provided additional antiviral protection.
51 Vero cells were also highly responsive to NSs siRNA. Similar to results seen in Soldan e t al. (2005) Levin et al. (2006 ), and Flusin et al. (2012), viral replication in Vero cells was significantly i nhibited early in infection, as titer increased only about 0.5 log for the first 48 hpi, and remained significantly lower at later time points. However, by allowing infection to continue up to 120 hpi, our study suggests that antiviral RNAi merely delays the onset of infection, as viral titer increased dramatically after 48 hpi and eventually reached 10 5.63 TCID 50 /mL (or 4.26 X 10 5 TCID 50 /mL ) by 1 20 hpi. These data suggest that short interfering RNA targeting the overlapping open reading frames for TENV nucleoprotein and small segment nonstructural protein temporarily inhibits TENV proliferation. In addition to the protecting naked RNA from recogni tion and cleavage by RISC, the nucleoprotein has several functions in facilitating the replication cycle for viruses in the Bunyaviridae The nucleoprotein forms ribonucleoprotein complexes that induce and signal the transition between replication and tran scription by the viral polymerase, forms multimers with other copies of the nucleoprotein, and interacts with viral RNA, viral glycoproteins, and possibly cellular proteins, though the nature and function of these interactions are unclear (Leonard et al. 2 005, Eifan and Elliott 2009, Longhi 2009). Furthermore, the region of the S segment targeted by NSs siRNA in this study was found to be highly conserved among orthobunyaviruses, and was found to be necessary for nucleoprotein nucleoprotein interactions in Bunyamwera, another orthobunyavirus in the same serogroup as TENV (Leonard et al. 2005, Eifan and Elliott 2009). In light of the many putative functions of TENV nucleoproteins, it makes sense that antiviral RNAi that targets and effectively disrupts nucleo protein synthesis has been shown inhibit or delay bunyaviral replication.
52 Moreover, the NSs gene also lies within the target region for NSs siRNA. Soldan et al. (2005) suggest that the NSs protein suppresses cellular RNA silencing machinery in orthobunyavi ruses, which is the very network that our study induces to stop viral replication. Considering the interferon response is defective in Vero cells, reducing the expression of the viral NSs gene may rescue the antiviral RNAi response. It makes sense that det ection and downregulation of viral RNA translation by the RNAi response, which is active in vertebrate systems, remains a major antiviral response in the absence of an interferon response. Moreover, in HeLa cells, where TENV NSs purportedly antagonizes bot h the interferon response and the antiviral RNAi response, siRNA induced knockdown of the overlapping reading NSs/N region of TENV appears to effectively suppress TENV replication, as is apparent in light of the inhib i tion that NSs siRNA provides against T ENV infection beyond the general protection conferred by the nonsense siRNA. The persistence of antiviral RNAi therapy varies widely across the model system and the virus studied. Several studies have indicated that the degradation of siRNA and cell divis ion may dilute siRNA dose over time (Novina et al. 2002, Chen et al. 2004b). However, while there were no measures taken to examine whether NSs siRNA and miR K12 11 stability differed significantly across time, we found that RNAi appeared to persist in our cell culture system for 132 hours following single dose treatment with 50 pmol, or approximately 300 micromolar ( M ) miR K12 11 at 96.35% knockdown of the firefly luciferase target. However, statistical analysis of the knockdown response can only be considered borderline significant at 132 hpi (p = 0.052), likely because of the highly variable response in the no miR K12 11 control. Alkhalil et al. (2009) observed
53 viral gene knockdown for up to 7 days in Vero E6 cells transfected with 10 M of antiviral siRNA a considerably lower concentration than used here, and therefore there is some precedence for extended antivir al RNAi in Vero cells. However, the lack of efficacious knockdown at 132 hours post transfection would help explain why the differences in antiviral response of NSs siRNA did not remain statistically different from nonsense siRNA treatment up to 132 hours post transfection (i.e., 120 hours post infection) in Vero cells. Interestingly, despite successful inhibition by NSs siRNA at 72 hpi in Vero cells, TENV that emerged from NSs siRNA treated Vero cells following escape from silencing at 72 hpi typically di d not show any mutations at the siRNA target site, as we saw only one instance of a non target sequence in the 46 samples that were sequenced at 72 hpi. This suggests that mutations were not wholly responsible for evasion of the induced immune response, an d that alternate mechanisms are thus responsible for evasion of silencing. Viral titer in the supernatant increases even in the initial stages of infection in all treatment conditions, suggesting that siRNA inhibition is not 100% efficacious. Of the small sample of TENV sequenced from the supernatant of NSs siRNA treated Vero cells at 24 and 48 hpi (n = 10 for both), none showed differences from the NSs siRNA target sequence. It is possible that viral replication persists and eventually overwhelms the intra cellular antiviral response, even when RNAi is induced by preventative inoculation with siRNA to the virus, and that even at low levels, replicating virus may be able to antagonize the cellular antiviral response. Fitness of Escape P opulations Pairwise ana lyses of the growth rates of NSs siRNA and RdRp siRNA escape populations were not statistically different from virus grown in nonsense siRNA treated
54 or no siRNA treated cells. Furthermore, the titers of NSs siRNA and RdRp siRNA escape populations did not d iffer from the control infections in HeLa or C6/36 cells at any time point. While the titer of TENV from RdRp siRNA treated Vero cells was significantly lower than TENV from untreated cells in Vero cells at 24 hpi, this difference disappeared in all subseq uent samples taken. Moreover, RdRp siRNA did not reduce the infectivity of TENV any more than nonsense siRNA did at any time points. Therefore, our results suggest that the fitness of TENV collected from Vero cells at 120 hpi did not change as a result of siRNA selection in any of the three cell culture systems tested. Going into this study, we hoped to use siRNA selection to select for non target TENV variants or to select for or induce mutations that were deleterious to TENV fitness, in hopes that these v ariants might be less fit in other cell culture systems. However, our data suggests that RdRp siRNA did not affect TENV replication or fitness by any metrics we used. Conversely, where NSs siRNA did delay the onset of infection, our data suggest that mutat ions or selection for non wild type variants were not responsible for escape from silencing with NSs siRNA, and that TENV virions with the NSs siRNA target sequence persisted. Because NSs siRNA treatment did not appear to change the sequence or infectivity of emergent virus, it does not appear that we were able to truly test whether inducing mutations or selecting for non target viral variants would affect TENV fitness. There may be several possible explanations why targeted viral sequences persisted. Firs tly, there may be little diversity in the TENV target region, and hence there may not be a variety of non target variants to select from. To that end, deep
55 sequencing of TENV prior to the study may have identified sequences with more variation. Similarly, siRNA targeting other NSs sequences may have identified more mutable regions of the TENV genome. Furthermore, mutations that would abrogate NSs or nucleoprotein function may not have been viable or may have had lower replication rates than targeted TENV, i n which case only targeted variants would remain by 120 hpi. Maintenance of NSs integrity may be advantageous to TENV fitness in Vero, where NSs presumably antagonizes the antiviral RNAi. However, bunyaviral NSs is not required for efficient replication i n C6/36 cells (Blakqori et al. 2007, Szemiel et al. 2012), likely because of the dysfunctional RNAi response (Brackney et al. 2010, Scott et al. 2010). By applying siRNA selection pressure in C6/36 cells, where there may not be positive selection for the m aintena nce of NSs, TENV may have been more susceptible to selection for non consensus sequence variants in the C6/36 cell culture system. Presumably, we may have been able to induce or to select for variants in C6/36 cells that were less viable when re int roduced to immune competent cell systems. However, our attempts at siRNA induced antiviral silencing in C6/36 cells were unsuccessful, so this possibility could not be examined here. Incomplete silencing may also have been responsible for allowing siRNA t argeted TENV to escape silencing and persist in fresh cells. Berkhout and Das (2012) suggest that wild type virus will always outcompete other escape mutants in the absence of siRNA selection. Indeed, this corroborates findings by Gitlin et al. (2005), whe re RNAi resistant poliovirus that differed from wild type variant only maintained a fitness advantage over the wild type when siRNA selection was in place. NSs siRNA
56 and RdRp siRNA were designed according to the consensus sequence of TENV, and therefore, i ncomplete silencing would allow virions with the consensus sequence to escape siRNA selection and outcompete less fit variants. Holz et al. (2012) demonstrated that mutations in morbillivirus subjected to several rounds of RNAi sel ection were not evident u ntil as many as 20 serial passages through siRNA treated cells, suggesting that extended exposure to antiviral RNAi selection beyond the timeframe tested in our study may eventually induce mutations or select for non target variants. Even though we did n ot see mutations in siRNA target region, no further tests were conducted to examine whether siRNA selection caused other changes to viral populations that were not apparent in sequencing (e.g., change of particle to infectivity ratio, induction of formatio n of defective interfering particles, etc.). Conclusions The research herein used Tensaw virus (Family Bunyaviridae Genus Orthobunyavirus) as a model system for cell culture experiments examining the effects of inducing the antiviral RNA interference res ponse in vitro on the infectivity and genome of an arbovirus. While one TENV specific siRNA did delay the onset of inf ection in Vero cells, the siRNA sequences chosen did not induce or select for mutations that affected the viability of the virus that emer ged from siRNA selection, nor provides valuable insight into the mechanisms underlying how arthropod borne viruses evade the immune response and suggests future avenues for research into antiviral RNAi. For instance, it was supposed in many other bunyaviral studies that an initial reduction in titer was indicative of successful knockdown. However, our results suggest
57 that successful knockdown is marked by prolonged suppre ssion of viral replication, and that even successfully targeted by RNAi at first can eventually overcome treatment. This suggests that factors other than target site mutations and sel ection for non target variants need to be explored thoroughly. Similarly, a thorough examination of transcript levels, protein expression, and enzyme activity involved in antiviral RNAi in the face of virus mediated suppression of the immune system by bunyaviral NSs protein expression in Vero or HeLa cells would uncover the tim ing and the interplay in the antagonistic host pathogen relationship. F uture efforts should also examine whether using different siRNAs or multiple siRNAs will result in sustained, efficacious knockdown of bunyavirus production, as has been seen in other s ystems using siRNA therapies against bunyaviruses. Hepatitis C, and HIV (Henry et al. 2006, ter Brake et al. 2006, Kumar et al. 2008, Shah et al. 2011, Flusin et al. 2012). Furthermore, Flusin et al. (2012) demonstrated that siRNA therapy is enhanced when combined with treatment with ribavirin, a guanosine analogue that also inhibits bunyaviral replication in vivo and in vitro (Watts et al. 1989, Bente et al. 2010). This suggests that where single dose regimens of siRNA do not work alone, combination with o ther antiviral treatments may make RNAi therapy a reality.
58 Figure 2 1. Adjusted f irefly l uciferase r atios In order to confirm that RNAi induced knockdown is sustained,Vero cells were co transfected with three plasmids (one expressing firefly lucif erase, one expressing Renilla luciferase, and an empty vector to equalize the volume transfected). In one condition, miR K12 11, which suppresses the expression of firefly luciferase, was also transfected. The ratio of firefly luciferase to Renilla lucifer ase was measured at 132 hours post K12 was adjusted to 1 to show differences as a measure of relative light units. Error bars indicate standard error. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 No K12-11 miRNA With K12-11 miRNA Relative Light Units Adjusted Firefly Luciferase Ratios
59 Table 2 1 Growth of Tensaw Passage 1 ( TENV P1 ) isolates from siRNA treated Vero cells Hours Post Infection TENV P1 NSs TENV P1 RdRp TENV P1 Nonsense No siRNA 0 1.50 1.50 1.50 1.50 24 1.65 a 1.59 b 2.57 b 3.00 b 48 2.06 a 3.36 ab 3.57 bc 4.61 c 72 4.02 a 4.50 ab 5.15 bc 5.33 c 96 5.15 a 5.8 8 a 6.29 ab 7.31 b 120 5.63 a 6.69 ab 6.58 bc 7.18 c Vero cells were pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) prior to infection with TENV FL06 at MOI of 0.01. Infectivity was measured by analysis of the T CID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. Error bars indicate standard error.
60 Figure 2 2 Growth of T ENV P1 isolates obtained after infecting short interfering (siRNA) treated Vero cells. Vero cells were pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) prior to infection with TENV FL06 at MOI of 0.01. Infectivity w as measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. Error bars indicate standard er ror. See Table 2 1 for values. 0 1 2 3 4 5 6 7 8 0 24 48 72 96 120 144 Titer (log TCID 50) Time (hpi) Growth of TENV P1 from siRNA treated Vero cells TSV-P1-NSs TSV-P1-RdRp TSV-P1-Nonsense No siRNA
61 Table 2 2 Growth of TENV P1 isolates from siRNA treated HeLa cells Hours Post Infection TENV P1 NSs TENV P1 RdRp TENV P1 Nonsense No siRNA 0 2.09 1.86 1.91 2.15 24 2.75 a 3.05 b 3.17 b 4.28 c 48 2.78 a 3.39 b 3.61 b 4.99 c 72 2.60 a 3.79 ab 3.88 b 6.05 c 96 3.27 a 4.16 b 4.41 b 6.69 c 120 3.50 a 4.18 b 4.30 b 5.93 c HeLa cells w ere pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) prior to infection with TENV FL06 at MOI of 0.01. Infectivity was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. Error bar s indicate standard error.
62 Figure 2 3 Growth of TENV P1 isolates obtained after infecting siRNA treated HeLa cells. HeLa cells were pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) prior to infection with TENV FL06 at MOI of 0.01. Infectivity was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time poin t. Error bars indicate standard error. See Table 2 2 for values. 0 1 2 3 4 5 6 7 8 0 24 48 72 96 120 144 Titer (log TCID 50) Time (hpi) Growth of TENV P1 from siRNA treated HeLa cells TSV-P1-NSs TSV-P1-RdRp TSV-P1-Nonsense No siRNA
63 Table 2 3 Growth of TENV from siRNA treated Vero cells in Vero cells Hours Post Infection TENV (P1V NSs 120) P2V TENV (P1V RdRp 120) P2V TENV (P1V nonsense 120) P2V TENV (P1V no siRNA 120) P2V 0 1.83 1.54 1.58 1.71 24 2.74 a 2.86 ab 2.57 ab 3.76 b 48 4.55 a 4.28 a 4.73 a 5.17 a 72 5.93 a 5.84 a 5.93 a 5.67 a 96 6.93 a 6.72 a 5.90 a 5.22 a 120 6.61 a 7.15 a 6.34 a 5.50 a TENV from Vero cells that were pre treated with NSs si RNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) was collected at 120 hpi, and diluted to MOI of 0.01 for infection of fresh Vero cells. Infectivity was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at th e time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. (P1V ____ 120) indicates the siRNA treatment for the first passage of TENV in Vero cells. Error bars indicate standard error.
64 Figure 2 4 Growth of TENV from siRNA treated Vero cells in Vero cells TENV from Vero cells that were pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) was collected at 120 hpi, and diluted to MOI of 0 .01 for infection of fresh Vero cells. Infectivity was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. (P1V ____ 120) indicates the siRNA treatment for the first passage of TENV in Vero cells. Error bars indicate standard error. See Table 2 3 for values. 0 1 2 3 4 5 6 7 8 0 24 48 72 96 120 144 Titer (log TCID 50) Time (hpi) Growth of TENV from siRNA treated Vero cells in Vero cells TENV (P1V NSs 120) P2V TENV (P1V RdRp 120) P2V TENV (P1V nonsense 120) P2V TENV (P1V no siRNA 120) P2V
65 Table 2 4 Growth of TENV from siRNA treated Vero cells in HeLa cells Hours Post Infection TENV (P1V NSs 120) P2H TENV (P1V RdRp 120) P2H TENV (P1V nonsense 120) P2H TENV (P1V no siRNA 120) P2H 0 1.83 1.76 1.72 1.92 24 3.07 a 3.33 a 3.46 a 3.82 a 48 4.37 a 5.54 a 5.25 a 5.39 a 72 5.87 a 6.24 a 6.63 a 6.23 a 96 5.74 a 6.7 8 a 7.05 a 6.75 a 120 5.97 a 6.57 a 6.73 a 6.58 a TENV from Vero cells that were pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) was collected at 120 hpi, and diluted to MOI of 0.1 for infection of fresh HeLa cel ls. Infectivity was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. (P1V ____ 120) indicates the siRNA treatment for the first passage of TENV in Vero cells. Error bars indicate standard error.
66 Figure 2 5 Growth of TENV from siRNA treated Vero cells in HeLa cells TENV from Vero cells that were pre treated with NSs siRNA, RdRp siRN A, nonsense siRNA or were not transfected (no siRNA) was collected at 120 hpi, and diluted to MOI of 0.1 for infection of fresh HeLa cells. Infectivity was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points i ndicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. (P1V ____ 120) indicates the siRNA treatment for the first passage of TENV in Vero cells. Error bars indicate standard error. See Tabl e 2 4 for values. 0 1 2 3 4 5 6 7 8 0 24 48 72 96 120 144 Titer (log TCID 50) Time (hpi) Growth of TENV from siRNA treated Vero cells in HeLa cells TENV (P1V NSs 120) P2H TENV (P1V RdRp 120) P2H TENV (P1V nonsense 120) P2H TENV (P1V no siRNA 120) P2H
67 Table 2 5 Growth of TENV from siRNA treated Vero cells in C6/36 cells Hours Post Infection TENV (P1V NSs 120) P2C TENV (P1V RdRp 120) P2C TENV (P1V nonsense 120) P2C TENV (P1V no siRNA 120) P2C 0 1.50 1.60 1.80 1.94 24 4.12 a 2.93 a 3.21 a 3.27 a 48 3.53 a 3.53 a 3.60 a 4.20 a 72 3.94 a 4.38 a 5.44 a 4.84 a 96 5.67 a 5.47 a 6.05 a 6.15 a 120 6.18 a 6.15 a 6.91 a 6.86 a TENV from Vero cells that were pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not tran sfected (no siRNA) was collected at 120 hpi, and diluted to MOI of 0.1 for infection of fresh C6/36 cells. Infectivity was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after dat a points express levels of statistical difference within a data set for that time point. (P1V ____ 120) indicates the siRNA treatment for the first passage of TENV in Vero cells. Error bars indicate standard error.
68 Figure 2 6 Growth of TENV from siRN A treated Vero cells in C6/36 cells TENV from Vero cells that were pre treated with NSs siRNA, RdRp siRNA, nonsense siRNA or were not transfected (no siRNA) was collected at 120 hpi, and diluted to MOI of 0.1 for infection of fresh C6/36 cells. Infectivit y was measured by analysis of the TCID 50 /mL values of tissue culture fluids collected at the time points indicated (hpi). Letters after data points express levels of statistical difference within a data set for that time point. (P1V ____ 120) indicates the siRNA treatment for the first passage of TENV in Vero cells. Error bars indicate standard error. See Table 2 5 for values. 0 1 2 3 4 5 6 7 8 0 24 48 72 96 120 144 Titer (log TCID 50) Time (hpi) Growth of TENV from siRNA treated Vero cells in C6/36 cells TENV (P1V NSs 120) P2C TENV (P1V RdRp 120) P2C TENV (P1V nonsense 120) P2C TENV (P1V no siRNA 120) P2C
69 APPENDIX A DETERMINING VIRAL TITER BY MEDIAN TISSUE CULTURE INFECTIOUS DOSE (TCID 50 / MILLI L ITER ) Modified from Reed and Muench (19 (2007) All cells were be maintained at 37C wi t hout carbon dioxide ( CO 2 ). 15 media containing 5% fetal bovine serum ( FBS ) and 50 micrograms per milliliter ( g/mL ) gentamycin will be used for cell mainte nance and virus dilution. 1. Calculate correct number of 96 cluster well (cw) plates needed for the titration. Up to eight rows of serial dilutions can be titered in each 96 cw plate with ten replicates per row. 5 X 10 3 cells will be seeded in each well in 5 0 microliters ( L ) of media. Therefore, to achieve this concentration, prepare 6 X 10 5 cells in 6 mL of media. Grow enough Vero cells accordingly. Each T 25 flask holds approximately 5 X 10 6 Vero cells at confluency. 2. Twenty four hours prior to infection, t rypsin ize the Vero cells, add one mL media for each mL trypsin used to loose cells. Determine concentration with hemocytometer. 3. Seed cells in 96 cw well with repeat pipettor. 4. Prepare 1/10 dilutions by filling sterile Starstedt tubes with 1.8 mL of media. 5. Pipet 200 microliters ( L ) of initial supernatant into the first tube with a P 200 pipettor. This is the 10 1 dilution. Make serial dilutions with 200 L of each tube per dilution up to 10 8 as needed. 6. Once dilutions are made for all time points and cond itions, inoculate ten wells with 100 L of the most diluted dilution using the repeat pipettor. Repeat for each stronger dilution until the highest dilution is reached for that condition. Repeat this step for all treatments and time points, using a differe nt disposable repeat pipettor tip per dilution set. 7. Once all cells have been inoculated, use sterile 5 milliliter ( mL ) pipettor to add two drops of sterile mineral oil to each well to prevent desiccation. Incubate cells for one week at 37C. 8. Examine cells under microscope. Identify wells where virus caused observable cytopathologic effects (CPE) and mark the well with X.
70 9. Calculate viral titer in a 96 cluster well as follows (see Figure A 1) : Reed and Muench equation: TCID 50 /mL = 10 A + 10 B/C + 10 1 + 10 0. 5 A = row where CPE is seen in all inoculated wells B = number of infected wells below the row with cytopathologic effects ( CPE ) in every well (i.e., after the row used to calculate variable A) C = number of wells inoculated for each dilution (i.e., 10 i n the above example) 10 1 = correction for the dilution. (i.e., 100 is used for every dilution, whereas TCID 50 is calculated in 1 mL) 10 1/2 = correction based upon Poisson distribution of units of infective particle per well Calculation of above example: TCID 50 /mL = 10 A + 10 B/C + 10 1 + 10 1/2 = 1 0 5 + 10 6/10 + 10 1 + 10 1/2 = 10 7.1 or 1.26 X 10 7
71 Row number (Dilution) Inoculated Wells Uninfected 1 2 3 4 5 6 7 8 9 10 11 12 Row 1 (10 1 ) X X X X X X X X X X Row 2 (10 2 ) X X X X X X X X X X Row 3 (10 3 ) X X X X X X X X X X Row 4 (10 4 ) X X X X X X X X X X Row 5 (10 5 ) X X X X X X X X X X Row 6 (10 6 ) X X X X X Row 7 (10 7 ) X Row 8 (10 8 ) Figure A 1. Graphical representation of 96 cluster well ( cw ) plate infected. X indicates a well with visible cytopathologic effects ( CPE )
72 APPENDIX B LUCIFERASE DEREPRESSION ASSAY I Luciferase Transfection and Expression 1. Seed Vero cells at 6 X 10 4 cells per 24 cluster well at 24 hours before transfection. 2. Co transfec t 2 nanongram s ( ng ) of pCMV Renilla luciferase vector (Promega), 20 ng of the Firefly luciferase pGL3 reporter construct (Promega), and 200 ng of an empty pcDNA 3.1/V5/HisA in triplicate (e.g., technical replicates) for 24 hours using Lipofectamine LTX ( Lifetech) transfection reagent per instructions. Additionally, for the treatment condition, transfect those three plasmids as well as 50 picomoles ( pmol ) of K12 11 microRNA mimic in triplicate. 3. After 24 hours, replace the transfection media with fresh medi a. 4. Salt Solution to remove trace amounts of media. 5. Place 100 microliters ( ) of Passive Lysis Buffer (Promega) into each well, and gently rock the cells for 15 minutes. 6. Scrap cells with pipet tip to facilitate detachment and lysis, and transfer cell containing buffer to 1.5 milliliters ( mL ) microcentrifuge tube. 7. Vortex each tube for 10 seconds. To remove excess cellular debris, centrifuge tubes at 12,000 rotations per minute ( rpm ) for 1 minute, and transfer supernatant to fresh 1.5 mL microcentrifuge tube. Freeze at 70 C until quantitation steps. II Luciferase Quantitation 1. Thaw cells prior to quantitation. Meanwhile, have the microplate luminometer program ready to measure the samples, because the respective luciferase signals decay quickly after the Luciferase Assay Reagent and the Stop & Glo Buffer are added. Measure lumi nescent signal for each luciferase for 24 intervals at 1 second per interval. 2. Add 20 of tube of the cell containing buffer to a clean well in the 96 well microplate. Add 100 of 1X Luciferase Assay Reagent to the cells. 3. Immediately place microplate i n the luminometer reader to measure firefly luciferase luminescence signal in each well. 4. Remove microplate from reader, and add 100 of Stop & Glo Buffer to quench the firefly luciferase reaction and complete the activation the Renilla luciferase signa l.
73 5. Immediately place microplate in the luminometer reader to measure the Renilla luciferase luminescence signal in each well. 6. Remove the first two and the last two measurement intervals from the calculation of each luminescent signal. The computer will th en take the average measurement of the remaining 20 intervals. III Calculation of Relative Light Units and Example Equation 1. Normalize the signal of the firefly luciferase expression signal by dividing the value of each replicates of the firefly luciferase signal by the value of each replicate from Renilla luciferase signal, for each technical replicate in all control and treatment samples. This will yield a value for the relative firefly luciferase signal for each technical replicate. 2. Calculate the average firefly luciferase signal for the control and the treatment samples by taking the average of the three technical replicates. Adjust the control value to a relative light unit of 1 in order to determine the relative knockdown of the firefly luciferase in t he treatment condition as a percentage.
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87 BIOGRAPHICAL SKETCH Daniel Mark Fitzpatrick was born in 1984 in Naples, FL, and at age 6 his family reloca ted to Gainesville H e knew from a young age that he would b e a member of the Gator Nation, and i n 2006, he graduated from the University of Florida with a dual Bachelor of Science degree in m icrobiology and p sychology. After a brief stint a baker and a waiter, Daniel took a research and development internship at the biotechnology company Regeneration Technologies Inc. in Alachua, FL, and rekindled his love for biological research. Dan iel started graduate schoo l in Insect Virology lab at the University of Florida Entomology and Nematology Department. He received his Master of Science in 2013.