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Proteolytic processing of the n-terminus of citrus tristeza virus open reading frame 1

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Proteolytic processing of the n-terminus of citrus tristeza virus open reading frame 1
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Vãzquez-Ortiz, Jorge, 1970-
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viii, 79 leaves : ill. ; 29 cm.

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Amino acids ( jstor )
Closterovirus ( jstor )
Enzymes ( jstor )
Genomes ( jstor )
In vitro fertilization ( jstor )
Open reading frames ( jstor )
Polyproteins ( jstor )
Proteins ( jstor )
RNA ( jstor )
Virology ( jstor )
Dissertations, Academic -- Plant molecular and Cellular Biology -- UF ( lcsh )
Plant molecular and Cellular Biology thesis, Ph.D ( lcsh )
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theses ( marcgt )
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Thesis (Ph.D.)--University of Florida, 2001.
Bibliography:
Includes bibliographical references (leaves 70-78).
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Printout.
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Vita.
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by Jorge Vázquez-Ortiz.

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PROTEOLYTIC PROCESSING OF THE N-TERMINUS OF CITRUS TRISTEZA
VIRUS OPEN READING FRAME 1












By

JORGE VkZQUEZ-ORTIZ


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2001






























To my loving family














TABLE OF CONTENTS
Page

LIST O F TABLES ....................................................... v

LIST O F FIG URES ..................................................... vi

A B STRA C T ........................................................... vii

1. IN TRO D U CTIO N ................................................. 1

Citrus Tristeza Virus: Classification and Genome Structure ........... 2
Viral Expression Strategies .................................... 6
Closterovirus Expression Strategy ............................... 7
Virus Population Structure .................................... 10
Different Types of Proteases are Encoded in Viral Genomes ......... 12
Viral Cysteine Proteases: Leader versus Main Proteases ............ 13
Closterovirus Protease Classification ........................... 16
O bjectives ................................................ 17

2. SEQUENCE ANALYSIS AND DETERMINATION OF
AUTOPROTEOLYTIC ACTIVITIES OF CITRUS TRISTEZA VIRUS
PUTATIVE CYSTENE PROTEASE DOMAINS ....................... 18

Closterovirus Genomes Encode for Papain-Like Cysteine Proteases ... 18
M aterials and M ethods ....................................... 23
V irus Isolate ......................................... 23
RNA Isolation and Complementary DNA (cDNA) Synthesis .. 23
Polymerase Chain Reaction. Amplification of Protease
Domains and Cloning Strategy .................... 23
In vitro Transcription and Translation ..................... 25
Protease Inhibitor Assays ............................... 26
R esults ................................................... 26
CTVL 1 is Proteolytically Active ......................... 26
No Proteolytic Activity was Detected for the CTVL2 Protein .. 27
Processing Pattern of the N-terminal Region of CTV ORF 1
Revealed Three Cleavages in the Region ............ 30
Sensitivity to Protease Inhibitors ......................... 30
D iscussion ................................................ 32











3. SITE DIRECTED MUTAGENESIS OF PUTATIVE ACTIVE AND
CLEAVAGE SITES OF CTVL 1 AND CTVL2: THE N-TERMINAL
PROCESSING DYNAMICS ........................................ 36

Materials and Methods ...................................... 38
Site Directed Mutagenesis .............................. 39
In vitro Transcription and Translation ..................... 40
Results ................ .............................. 40
Cysteine 403 and Histidine 1 are Involved in CTVL I Activity.. 40
CTVL 1 Might be Responsible for More Than One Cleavage
Within the CTV ORFI N-Terminal Region .......... 41
CTVL2 is an Active Protease ........................... 42
CTVL 1 Might be an Additional Substrate for CTVL2 ........ 42
Mutations at the P1 Position of PCS1 and PCS2 are not Well
Tolerated ..................................... 45
D iscussion ................................................ 46

4. CIS TRANS ACTIVITY OF CTVL I AND CTVL2 .................... 54

M aterials and M ethods ....................................... 57
Virus Isolate, cDNA Synthesis and Cloning ................ 57
Post-Translational Cis/Trans Activity Assays ............... 57
Co-Translational CisiTrans Activity ...................... 57
R esults ................................................... 58
CTVL 1 Encodes a Cis Acting Protease Tested in an
In vitro A ssay ........................................ 58
CTVL 1 -L2-PCS2 did not Complement the Proteolytic Reaction
in a Post-Translational Assay ..................... 59
CTVL2 may have Trans Activity as Shown by a Co-Translational
Trans Proteolytic Assay .......................... 62
D iscussion ................................................ 65

5. SUMMARY AND CONCLUSIONS ................................. 69

6. REFEREN CES .................................................. 70

7. BIOGRAPHICAL SKETCH ........................................ 79














LIST OF TABLES


Table Page

2.1: Sequence of oligonucleotides used to amplify PCP domains from ORF 1 of
CTV strain T2K .................................................. 25

3.1: Oligonucleotide primer sequences used to introduce mutations to the putative
active sites ...................................................... 39

3.2: Oligonucleotide primer sequences used to introduce mutations into the
putative cleavage sites ............................................. 40













LIST OF FIGURES


Figure Page

* 1.1: Citrus tristeza virus symptoms in different host species ................ 3

* 1.2: Schematic representation of citrus tristeza virus genome organization and
expression strategies ............................................... 5

0 1.3: Electron micrographs of citrus tristeza closterovirus particles ............ 7

S 1.4: General mechanism of action of cysteine proteases ................... 14

0 2.1: Protein sequence analysis of described and putative cysteine proteases of
Potyvirus and Closterovirus ......................................... 22

0 2.2: Schematic representation of CTV ORF I N-terminal region and expression
constructs of CTV PCPs ........................................... 24

* 2.3: Analysis of proteolytic activity of the N-terminal region of CTV ORF1... 29

* 2.4: Effect of chemical protease inhibitors in the proteolytic activity of CTVL 1
and CTV L2 ..................................................... 31

0 3.1: Site directed mutagenesis of predicted catalytic amino acids of CTVL 1 and
C TV L2 ........................................................ 44

* 3.2: Site directed mutagenesis of PCS 1. Localization of CTVL2 substrate site 44

0 3.3: SDS-PAGE autoradiogram of CTVL1-L2-PCS2 proteins mutagenized at the
P 1 position of their putative cleavage sties ............................. 46

0 4.1: CTVL1-PCS 1 trans complementation of proteolytic activity ........... 60

* 4.2: CTVLI-L2-PCS2 tans proteolytic activity .......................... 61

0 4.3: Assessment of cis-trans activity of CTVL 1 and CTVL2 .............. 64














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PROTEOLYTIC PROCESSING OF THE N-TERMINUS OF CITRUS TRISTEZA
VIRUS OPEN READING FRAME 1

By

Jorge Vdzquez-Ortiz

August, 2001


Chairperson: Charles L. Niblett
Major Department: Plant Molecular and Cellular Biology Program

Citrus tristeza virus (CTV) causes one of the most economically important

diseases in commercial citrus worldwide. CTV is a member of the genus Closterovirus in

the Closteroviridae family of positive stranded plant RNA viruses. It is characterized by

long flexuous rod-shaped particles, with a genome of- 19 Kb with 12 open reading

frames (ORFs). ORF1 encodes a polyprotein with two putative papain-like cysteine

protease domains (PLPs) designed as CTVL1 and CTVL2, a helicase, a methyl

transferase and a RNA dependant RNA polymerase (RdRp) expressed by a +1 ribosomal

frameshift.

In the present study, the N-terminal region of ORF1 was cloned and used as a

template in a transcription and translation assay. The presence of two cysteine proteases

was demonstrated in vitro. The putative catalytic amino acids for both cysteine protease









domains of CTV were previously identified by sequence alignment with the papain-like

protease of beet yellows virus. Site directed mutagenesis confirmed C3 H596 and C896 -

H-16 as the residues in the active sites for CTVL1 and CTVL2, respectively. Proteolytic

processing of the region includes a unique cleavage by the CTVL1 domain at the first

putative cleavage site (PCS) G484-G485, located at its C-terminus. A second cleavage is

mediated by CTVL2 at the second PCS G976-G977. These proteolytic activities released

both proteases from the polyprotein, and they were independent of each other. An

additional cleavage is mediated by CTVL2 at a previously unreported within the first

protease. This second activity of CTVL2 is dependent on CTVL1 activity. All the

proteolytic activities were insensitive to a protease inhibitor cocktail of broad specificity

for serine, cysteine and aspartic proteases.

Under the conditions tested, we were not able to detect trans activity for CTVLl;

however, we obtained some evidence, which indicates that CTVL2 may be able to act in

trans at the newly detected cleavage site. A working model includes independent

autocatalytic releases of CTVL1 and CTVL2 from the polyprotein and a trans cleavage of

CTVL1 mediated by CTVL2. The biological significance of the processing of the ORFI

N-terminal region remains to be determined.














CHAPTER 1
INTRODUCTION

Citrus tristeza closterovirus (CTV) causes "tristeza," the most economically

important viral disease of citrus (Kitajima et al., 1964; Rocha-Pefia et al., 1995).

"Tristeza," which means sadness in Spanish, describes a decline disease caused by CTV

and occurs on citrus scions that are propagated on sour orange rootstocks (Bar-Joseph et

al., 1989). Sour orange rootstock has been widely used in the citrus industry due to the

tolerance of this rootstock towards different pathogens such as Phytophtora (Klotz,

1978), and several graft-transmissible pathogens. Also, sour orange rootstocks are

adaptable to different soils and are compatible with most citrus (Rocha-Pefia et al., 1995).

Citrus tristeza virus causes a variety of symptoms, depending both on the infecting

strain as well as the infected host/rootstock combination (Rocha-Pefia et al., 1995).

Symptoms include quick decline on sour orange rootstock (Figure 1. IA), honeycombing

at the bud union (Figure 1.1 B), stem pitting of sensitive cultivars (Figure 1.1 C), leaf vein

corking of sensitive cultivars (Figure 1.1 D), reduced fruit size and quality (Figure 1.1 E)

and vein clearing and leaf cupping (Figure 1.1F).

As an indicator plant, Mexican lime (Citrus aurantifolia (Christm.) Swingle) is

the most sensitive to CTV, but the severity of the symptoms does not necessarily

correlate with those observed in other hosts. Decline inducing (DI) strains are detected in

sweet orange (Citrus sinensis (L.) Osbeck) grafted onto sour orange seedlings. When










CTV-DI infected budwood is propagated onto sour orange (Citrus aurantium (L.))

seedlings, severe stunting results. Seeding yellows (SY) strains are identified on sour

orange, acid lemon (Citrus limon (L.) Burm), and grapefruit (Citrus paradisi (Macf.))

seedlings, which suffer chlorosis and stunting. For the strains which cause stem pitting

(SP) on grapefruit and/or sweet orange, there is an indexing system on Duncan grapefruit

and Madame Vinous sweet orange seedlings. CTV-SP-infected plants in the field show

longitudinal pits formed in the wood of the stems/branches of the scions, independent of

the rootstock. There is loss of plant vigor and yield reduction. Also vein clearing and

vein corking of leaves of sweet orange have been observed as symptoms for SP strains

(Rocha-Pefia et al., 1995).

Citrus Tristeza Virus: Classification and Genome Structure

CTV belongs to the order Nidovirales and is a member of the Closteroviridae

family of positive single-stranded RNA viruses according to the International Union of

Microbiology Societies classification (van Regenmortel et al., 2000).

The initial characterizations of the virus included the detection of thread-like

particles associated with the disease symptoms. These particles were about 2,000 pLm in

length and 10-12 Lm in diameter, and they resembled those of beet yellows virus

(Kitajima et al., 1964). The CTV genome was described as single-stranded positive sense

RNA (+ssRNA) by Bar-Joseph and Lee (1989) of approximately 20Kb in length (Bar-

Joseph et al., 1985). Once the complete genome of the T36 isolate was sequenced, it was

determined to have a 19,296 nt sequence, with 12 open reading frames (ORFs) that

potentially coded for 17 products (Pappu et al., 1994; Karasev et al., 1995).
















































Figure 1.1: Citrus tristeza virus symptoms in different host species. (A) Quick decline of
a sweet orange tree on sour orange rootstock. (B) Honeycombing of the bud union of a
sour orange rootstock. (C) Stem pitting in stems of Mexican lime seedlings. (D) Vein
corking symptoms on leaves of Mexican lime Citrus aurantifolia. (E) Grapefruit from a
tree infected with a stem pitting isolate of CTV; the fruit on the right comes from an
uninfected tree. (F) Vein clearing and cupping in leaves of Mexican lime.
Pictures taken from www.ecoport.org Pictures A, B and E by Lee, R. Pictures C, D, F by
Roistacher, C. N.











A diagram of the CTV genome with the putative open reading frames (ORFs) is

shown in Figure 1.2A and the expression strategies employed by CTV are shown in

Figure 1.2B. Sequence analysis allowed the assignment of potential functions for most of

the ORFs (Karasev et al., 1995). Currently, experimental evidence is being obtained to

corroborate some of these putative functions. The genome organization includes a 5'

proximal polyprotein of a calculated molecular mass of 349 KDa. This polyprotein

contains domains for two putative papain-like cysteine proteases (CTVLI and CTLV2), a

methyl transferase (MT), a helicase (HEL), and a RNA dependant RNA polymerase

(RdRp) that is expressed via a +1 ribosomal frameshift (;evik, personal communication)

that defines the ORFIa and ORFIb (Karasev et al., 1995).

The functions of genes located in the 3' region of the genome include a heat shock

protein 70 homolog (HSP70h) which has been detected by antibodies in CTV- (Rosales,

personal communication) and BYV- infected tissues (Napuli et al., 2000) by tissue

printings, as well as in association with the virion (Figure 1.3B). A duplicated coat

protein (p27) is expressed in CTV- infected tissues (Febres et al., 1994), and both the coat

protein (CP) and its diverged copy form part of the virion (Figure 1.2A; Febres et al.,

1996). This coat protein duplication is common in the Closteroviridae family, and it has

been suggested that the duplication occurred before the separation of BYV and CTV from

their common ancestor (Boyko et al., 1992).

Some of the functions of the 3' ORFs of closteroviruses have been elucidated

through the construction of a full length BYV cDNA infectious clone. That study

revealed that the gene product of the ORF 1 a/b is sufficient for RNA replication and











0k 10 15 20
II I

PRO-1 MMX HEL p33 p65 P27 p18 p20


PFD-2 RdRp p6 p61 CP p13 p23
01" la lb 2 3 4 5 6 7 8 910 11

B -






Viral cellular Ribosomal Subgenomic
proteases Pro teases? + 1 frameshift RNAs




Figure 1.2: Schematic representation of the citrus tristeza virus genome organization and
expression strategies. Panel A represents the open reading frames and the putative
proteins encoded. Panel B represents the genomic and sub genomic RNAs as single lines.
The putative proteins translated from each RNA are shown as boxes (Solid boxes
represent proteins of demonstrated activity or presence in CTV-infected tissue).
Figure from Manjunath et al., (2000).



transcription. This ORF la/b self-replicating clone was used as a vehicle to evaluate the

effects of adding specific 3' ORFs to the complex. These experiments revealed that the

ORF that encodes the p21 protein functions as an enhancer of genome amplification

(Peremyslov et al., 1998). Other functions have been revealed for other 3' ORFs, e.g. a 3'

end proximal ORF encodes a 23 kDa protein with the ability to cooperatively bind single-

stranded and double-stranded RNA in a non-sequence specific dependant manner (L6pez

et al., 2000). A protein, designated as p20, has been reported to interact strongly with










itself in a yeast two hybrid system and apparently forms amorphous inclusion bodies in

infected protoplasts, but no biological function has been assigned to it (Gowda et al.,

2000). The expression and function of the remaining ORFs in the CTV genome remain to

be identified and characterized.

Viral Expression Strategies

Among the positive-sense RNA viruses, a general classification into two big
"super groups," picorna- and alpha-like, can be made according to the expression

strategies used in the viral infection-replication cycle. The picoma-like viruses include

viruses in the Potyviridae, Comoviridae and Sequiviridae families. The genomes of these

viruses encode a long polyprotein that is proteolytically processed into smaller functional

domains by virus-encoded proteases. This expression strategy produces equimolar

amounts of all the viral proteins (Krdusslich and Wimmer, 1988). The expression of a

polyprotein allows the temporal and spatial control of the activity of the specific domains

as they become available in the form of individual proteins or processing intermediates

(Garcia et al., 1999).

The second class is comprised of the alpha-, como- and sobemo-like groups and

includes the Closteroviridae family. This group includes viruses that use a variety of

expression strategies. The synthesis of a polyprotein that is proteolytically processed into

non-structural proteins, including the RNA polymerase, mediates the synthesis of

transcripts known as sub-genomic RNAs (sgRNA). These sgRNAs encode the ORFs

downstream from the polyprotein, and these include the viral structural proteins. This

strategy allows the differential expression of enzymatic non-structural proteins versus the











A B

















Figure 1.3: Electron micrographs of citrus tristeza closterovirus particles. (A) Viral
particle immunolabeled with a gold-conjugated rabbit polyclonal antiserum against the
diverged copy of the coat protein, showing its localization at one end of the particle. (B)
Viral particle immunolabeled with a gold-conjugated chicken polyclonal antiserum
against the HSP7Oh protein, showing its interaction with the viral particle. Gold labeled
particles are shown with an arrow.
Picture A by Febres, V. (1996). Picture B by Rosales, M. (2001).

increased need for structural proteins (Krausslich and Wimmer, 1988). Polyprotein

expression is frequently combined with other strategies such as alternative translation

initiation sites, frameshifting, and readthrough of suppressible termination codons (Garcia

et al., 1999). The low frequency with which these alternative mechanisms occur

represents an additional mechanism to control the differential expression of different

types of proteins (KrAusslich and Wimmer, 1988).

Closterovirus Expression Strategy

Sequence analysis of the BYV genome revealed the presence of a polyprotein

encoded by the ORF ua/b (Agranovsky et al., 1994). The Closterovirus polyprotein

encodes one or two papain-like cysteine proteases, a MT and a HEL (Agranovsky et al.,











1994; Karasev et al., 1995; Jelkmann et al., 1997; Zhu et al., 1998). A dual activity has

been shown for the BYV L-pro. First, auto-proteolytic release from the polyprotein was

demonstrated in an in vitro transcription and translation assay (Agranovsky et al., 1994)

as well as in an in vivo assay, where it was demonstrated that the proteolytic release of L-

pro was essential for RNA replication (Peremyslov et al., 1998).

Analysis of BYV L-pro mutants revealed that this protein had a high tolerance to

structural changes in its N-terminal region, with the exception of a 54 amino acid stretch

at the 5' region of the ORF, which was important for virus viability. It also was

demonstrated that this protein was not essential for viral genome amplification, but its

activity increased the viral RNA level 1,000-fold when compared to the basal level

(Peremyslov et al., 1998; Peng and Dolja, 2000).

Further maturation of the closterovirus polyprotein has been determined by the

use of monoclonal antibodies against the BYV methyl transferase and helicase, which

have been detected as individual proteins in BYV-infected tissue (Erokhina et al., 2000).

Similarly, the presence of the RdRp as an individual protein in CTV-infected tissue has

been reported ((;evik, personal communication). The nature of the protease(s)

responsible for the release of these proteins from the polyprotein remains to be identified.

The expression of the polyprotein also includes a ribosomal + 1 frameshift, from

which the RdRp is expressed. This event has been experimentally demonstrated with

both in vivo and in vitro assays, and it was estimated to occur in 1-5 % of the translation

events ((;evik, personal communication). The remainder of the closterovirus ORFs are

likely to be expressed via 3'-co-terminal sgRNAs. The presence of six subgenomic RNA











species has been detected in BYV-infected tissues, whereas there is a great variability in

the number of sgRNA species detected in CTV-infected tissues (Agranovsky, 1996).

Studies on the kinetics of accumulation of CTV RNAs revealed temporal control of the

synthesis of the different sgRNAs both in host and non-host protoplasts (Navas-Castillo

et al., 1997).

This temporal control was further studied with the use of a BYV infectious clone

in which individual genes were tagged with bacterial P-glucuronidase (GUS). Analysis

of the results revealed that the temporal regulation of gene expression included early

expression of HSP70h, CP and its diverged copy, as well as the p21 protein, while the

expression of two other ORFs (p64 and p20) was related to the late phase of viral

infection. This study also revealed that the expression of the 3' ORFs can affect

transcription of sgRNA species since the deletion of six of the 3' ORFs resulted in the up

regulation of the remaining sgRNAs in the deleted construct. This pattern of temporal

regulation of multiple transcriptional units is unique among RNA viruses (Hagiwara et

al., 1999).

During infection with CTV, the presence of RNA molecules that consisted of

different deletions of the CTV genome and included variable portions of the 5'- and 3'-

terminal regions was observed (Mawasssi et al., 1995a; b). These defective RNA (D-

RNA) molecules seem to be replicated via a double-stranded RNA (dsRNA) intermediate

using a replicase-driven template switching mechanism (Ayll6n et al., 1999). The

presence of D-RNAs has been related to reduced accumulation of the helper virus,

inducing attenuation of symptoms in tomato bushy stunt virus-infected plants (Scholthof










et al., 1995) and to the exacerbation of symptoms in broad bean mottle virus infections,

according to the infected host (Romero et al., 1993). Their presence also has been found

not to interfere with symptoms in clover yellow mosaic virus- (White et al., 1991),

cucumber mosaic cucumovirus- (Graves et al., 1995), and CTV- (Ayll6n et al., 1999)

infected plants.

The presence of a D-RNA of almost identical sequence in two CTV isolates (T317

and T318) that greatly differed in their pathogenicity suggested that this D-RNA was not

the cause of the increased pathogenicity observed for the T318 isolate (Ayll6n et al.,

1999). Alternatively, D-RNAs with 5'- regions larger than 4,000 nt present in the CTV

isolate VT have been implicated in the suppression of seedling yellows symptoms on

specific hosts (Yang et al., 1999).

Virus Population Structure

The long term survival of viruses in their host (persistence) is affected by genetic,

ecological, and environmental factors. There are three mechanisms that describe the

persistence of a virus in its host. These include the following: balance between number

of infected cells and multiplication of the host cells; persistence in a limited number of

cell types; and continuous infection of a susceptible host with or without persistence in

infected cells (Domingo et al., 1998).

For the tristeza disease, the perennial nature and the susceptibility of citrus hosts

result in persistence of the virus in the infected plants. A consequence derived from this

is that the host is susceptible to multiple infections with the virus over the years, resulting

in a viral population that can be a mixture of different genotypes (Manjunath et al., 2000).










The presence of different genotypes in CTV-infected plants has been experimentally

demonstrated by the use of single-stranded conformational polymorphism (SSCP) of the

genes that encode the coat protein and its diverged copy. These experiments showed that

CTV isolates were composed of a population of genetically related variants (haplotypes),

having a predominant one in the population, although there was also a case of two

haplotypes with high divergence in the same isolate (Kong et al., 2000; Niblett et al.,

2000).

The presence of viral haplotypes in CTV isolates revealed the possibility of their

existence as quasispecies in the infected plant (Manjunath et al., 2000). Due to the

absence of proofreading activity of viral RNA polymerases (Drake and Holland, 1999),

RNA virus populations consist of complex distributions of genomes carrying different

mutations forming the population structure known as quasispecies (Domingo et al.,

1998). These quasispecies are represented by molecular variants of a genotype in the

range of I or 2 % difference between them (Davis, 1999).

The genetic organization of the viral population as a quasispecies represents an

adaptive strategy as it constitutes the raw material on which selective forces and random

sampling events act in the molecular evolution of RNA viruses (Domingo et al., 1998). A

study of sequence identity of five mild CTV isolates (asymptomatic in field trees and

causing only weak symptoms on the indicator plant, Mexican lime) from different

geographic and host origins revealed little variation among these isolates (Albiach-Marti

et al., 2000). That study reveals the importance of the interactions between specific viral

and host determinants. Most of the mutations among the five isolates were silent











mutations or changes that resulted in similar amino acids, suggesting that this CTV

genotype is well adapted to its hosts, and has not changed in several hundred years.

Another study of the haplotype distribution of CTV isolates after host change or aphid

transmission revealed changes in the populations of the original isolate and successive

subisolates. The extent of those changes was greater than that observed between isolates

from different geographical locations. These results suggested that adaptation to a new

host changed the haplotype distribution, this change being more important than the

geographical origin of the isolate (Ayll6n et al., 1999) even though this is not a constant

outcome of the adaptation process.

Different types of Proteases are Encoded in Viral Genomes.

Proteases are encoded in the genomes of diverse viral groups, which include non-

enveloped single-stranded RNA (ssRNA) viruses, enveloped ssRNA viruses, non-

enveloped double-stranded DNA (dsDNA) viruses and enveloped dsDNA viruses (Bab6

and Craik, 1997). Proteolysis of viral polyproteins is found primarily in positive-sense

(+) ssRNA viruses and retroviruses (Krdusslich and Wimmer, 1988).

Various studies have revealed several common characteristics for the proteases of

this group (Ryan and Flint, 1997 and references therein). They are commonly found as a

domain of a larger protein, which can represent alternative processing products, and their

activity can depend on the specific processing, intermediate location, and also can be

modified by interaction with other proteins or RNA. Viral proteases also can cleave host

proteins in trans, thus modifying host functions and can be regulated until a particular

cellular environment is encountered (Ryan and Flint, 1997).









13

Cellular proteases have been classified according to their active site nucleophiles

into serine-, cysteine-, aspartyl- and metallo- proteases (Ryan and Flint, 1997). Serine-

proteases are characterized by having an aspartate, a histidine and a serine as a catalytic

triad (Ryan and Flint, 1997). Cysteine-proteases have a catalytic dyad of cysteine and

histidine. On the carboxyl-side of the catalytic cysteine, there is a conserved aromatic

residue that is characteristic of all papain-like cysteine-proteases (Ziebuhr et al., 2000).

The aspartic or acid proteases are characterized by the presence of two aspartic acid

residues in their active site, and the metallo-proteases perform their nucleophilic attack

using a metal cation (Ryan and Flint, 1997). The general mechanism of catalysis by

cysteine proteases is through an acid-base formation of an acyl-thiol intermediate

followed by a hydrolysis reaction (Figure 1.4).

The use of chemical inhibitors has helped in the classification of the proteases. As

examples, aspartic-proteases are specifically inhibited by pepstatin; cysteine-proteases by

trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) and cystatins; metallo-

proteases by chelating agents and seine proteases by diisopropyl fluorophosphate and

phenylmethylsulphonyl fluoride (Kay and Dunn, 1990).

Viral Cysteine Proteases: Leader versus Main Proteases

The presence of papain-related cysteine proteases has been reported for several +

ssRNA viruses. Protein alignments of the PLPs from different + ss RNA viruses has

revealed low conservation of the sequences of these proteases. Only the CW(Y)

dipeptide, which includes the catalytic cysteine, is conserved, and there is a much lower

conservation in the area surrounding the catalytic histidine.















_ IN,.
-I


R
I
C=0
I
H-N
I
R'


F S" *Him + RCOOH



3" P, "Him -
I
C-0

OH


R


S- HN.
R' HIM


S-C=o NH2 m
I Im
R'

HR0 R'-NH,
R



H H


Figure 1.4: General mechanism of action of cysteine proteases. The acyl-thiol
intermediate occurs by the formation of a non-covalent complex between the enzyme and
the substrate. This complex undergoes acylation, forming and releasing an amine R'-
NH2; this is followed by a deacylation step, which releases the second product,
regenerating the free enzyme. Im and +Him refer to imidazol and protonated imidazol,
respectively. (Rao et al., 1998).


This same protein alignment prompted the grouping of these viral proteases into

two classes; leader and main proteases (Gorbalenya et al., 1991). Leader proteases can

mediate a single cleavage at their own C-termini, or a double cleavage at sites located in

the amino-terminal half of the polyprotein. The cleavage site for leader proteases has at

least a small residue, and their active site is composed of a cysteine and a downstream

histidine. The main proteases represent proteins that mediate the processing of the non-











structural proteins, being in some cases the only protease encoded in the viral genome

(Gorbalenya et al., 1991; Ziebuhr et al., 2000). Sequence alignments revealed that the

main protease group possesses an additional conserved domain, the "X" domain

(Gorbalenya et al., 1991) and its activity was linked to the ability to perform trans-, but

not cis-, proteolytic cleavages for the rubella virus non-structural protease (Liang et al.,

2000).

Even though the sequence alignments provided some insight for the classification

of PLPs, experimental confirmations have not always been as straightforward. Rubella

virus (RUB) is an enveloped + ssRNA virus of the family Togaviridae. Part of its

genome encodes a polyprotein with an RdRp, a helicase and a papain-like cysteine

protease (RUB NSP). In vitro transcription and translation assays and site directed

mutagenesis allowed the identification of the catalytic amino acids as well as the residues

at a cleavage site. Co-expression of substrates and enzymes failed to complement the

reaction in trans, resulting in RUB NSP being reported only as a cis-acting protein. This

observation disagreed with the "main protease" prediction made by Gorbalenya et al.

(1991), but there were also characteristics such as the median location of the protease in

the polyprotein instead of N-terminal location and its post-translational, instead of co-

translational, maturation that also differed from the characteristics typical of leader

proteases (Chen et al., 1996).

Further research into the activity of RUB NS protease revealed that this protein

was able to perform trans-proteolysis (Yao et al., 1998), and it required the presence of

divalent cations for this activity. This protein was reclassified as a main protease, but











again, there were several characteristics such as its cleavage at a single site, and a space

of about 40 residues between the active site and cleavage site as for the typical leader

proteases, which did not fit with the description typical of main proteases. This suggested

that the classification of the viral papain-like protease family was more complicated than

initially thought (Liu et al., 1998).

The characterization of the metal ion binding activity of RUB NS protease

revealed the critical residues for the activity and showed that Zn> did not have a major

effect on the secondary structure of the protein. It also was shown that this proteolytic

activity could be blocked by metal ion chelators and the metallo-protease inhibitor

captopril. This finding suggested that RUB NS protease is not a papain-related cysteine

protease, but a novel metallo-protease (Liu et al., 2000).

Other proteases that have been classified as main proteases are the alphavirus

nsP2 protease (Hardy and Strauss, 1989), foot and mouth protease (Kirchweger et al.,

1994) and coronavirus PLP- I protease (Bonilla et al., 1997).

Closterovirus Protease Classification

The proteases of CTV have been classified in the MEROPS database as belonging

to the CA clan, family C42 (Rawlings and Barrett, 2000). Currently the two PLP of CTV

and the BYV L-pro are the only members of that family in the MEROPS database. BYV

L-pro is the type member of the C42 family. Sequencing of the BYV genome revealed

the presence of a putative papain-related cysteine protease and its activity was

corroborated in an in vitro transcription and translation assay (Agranovsky et al., 1994).










Objectives

The major objectives of this investigation are the characterization and study of the

proteolytic activities of the N-terminal region of CTV ORF la/b. This will contribute to a

better understanding of the biology of CTV and aid in the development of more effective

control measures. The specific objectives of this research are the following:

1. Determine the proteolytic activity ofCTVL I and CTVL2.

2. Determine their sensitivity to chemical protease inhibitors.

3. Determine the effect of site directed mutagenesis of the putative amino acids
on the active sites of both CTVL 1 and CTVL2.


4. Site directed mutagenesis of the amino acid that occupies the P 1 position for
both putative cleavage sites.


5. Determine potential cis and trans proteolytic activities of CTVL I and CTVL2
using CTV ORF I N-terminal region as substrate.


6. Establish a working model for the CTV ORF 1 N-terminal proteolytic
processing.













CHAPTER 2
SEQUENCE ANALYSIS AND DETERMINATION OF AUTOPROTEOLYTIC
ACTIVITIES OF CITRUS TRISTEZA VIRUS PUTATIVE CYSTEINE PROTEASE
DOMAINS


Closterovirus Genomes Encode for Papain-Like Cysteine Proteases

The presence of genes encoding classical cysteine proteases related to cellular

papain-like proteases has been reported in the genome of several positive-stranded RNA

viruses (Gorbalenya et al., 1991). Beet yellows closterovirus (BYV) is the type member

of the Closteroviridae, a group of plant viruses with flexible filamentous particles and a

single stranded (ss) positive sense (+) RNA genome.

Sequence analysis of the complete genome revealed similarities with the genomes

of Tobraviridae. This similarity allowed the identification of a helicase, a methyl

transferase and an RNA-dependant RNA polymerase (RdRp) domain within the first

open reading frame (ORF) of BYV, which was designated as the replication complex

(Agranovsky et al., 1994).

Alignments of the replication complexes of the Closteroviridae and Tobraviridae

viral groups reveal two unique regions for BYV (Closteroviridae) that account for the

difference of 1387 and 1328 residues with respect to tobacco rattle virus (TRV,

Tobraviridae) and pea early browning virus (PEBV, Tobraviridae) ORF I translation

products, respectively. These unique sequences did not produce any significant result

when compared against the database, but a motif analysis identified the sequence GLCY,










which resembles the sequences around the active site of papain-like thiol proteases

(Agranovsky et al. 1994).

The region that encodes the putative papain-like cysteine protease in the BYV

genome was cloned and used as a template for an in vitro transcription and translation

reaction (Agranovsky et. al, 1994). This experiment confirmed the presence of

proteolytic activity within the complex. The active amino acids involved in the

proteolytic activity were identified by site-directed mutagenesis and were mapped to the

conserved residues Cys509 and His569 as well as the cleavage site at Gly88-Gly59. Point

mutations of any of these amino acids resulted in the loss of proteolytic activity, whereas

mutations in non-conserved amino acids near the predicted active site had different

effects. Substitution of His556 was tolerated, while substitutions of Cys517 and Cys518

drastically reduced the proteolytic activity but did not abolish it completely (Agranovsky

et al., 1994).

Sequencing of the citrus tristeza virus (CTV) genome revealed the presence of

two putative papain-like cysteine proteases, named CTVL I and CTVL2 (Karasev et al.,

1995). Pairwise comparisons revealed similarity between the two putative cysteine

protease domains of CTV ORF I with the unique papain-like cysteine protease domain of

BYV. This allowed the prediction of the putative amino acids in the active sites as well

as the putative cleavage sites (Karasev et al., 1995). The positions of the putative

cleavage sites predict two proteins of 484 (CTVL 1) and 492 (CTVL2) residues. When

the complete amino acid sequence of each predicted protein was compared with that of

the BYV L-pro sequence, the putative cysteine protease domain was mapped to the C-











terminal end of each protein and encompassed approximately 30 % of that sequence.

Comparisons of upstream sequences ofCTVL1, CTVL2, BYV L-pro and little cherry

virus (LChV, Closteroviridae) did not produce any significant alignment among them

(Karasev et al., 1995; Jelkmann et al., 1997; Zhu et al., 1998). When compared, the

papain-like cysteine protease (PLP) upstream sequences of LChV and lettuce infectious

yellows virus (LIYV, Closteroviridae) additional conserved sequences, interrupted by

deletions or insertions of different lengths were found, but they were not identified with

any known domain (Jelkmann et al., 1997).

A protein sequence alignment of the C-terminal regions of BYV L-pro, CTVL 1,

and CTVL2 with the helper component protease (HC-pro) of seven different potyviruses

is shown in Figure 2.1A. In this alignment it can be observed that although there is little

conservation among the sequences of closteroviruses when compared to potyviruses, the

putative and already confirmed active amino acids are conserved in all the proteins. The

sequences of BYV L-pro vs. CTVL1 showed a 14 % identity and 25 % similarity; BYV

L-pro vs. CTVL2 showed 16 % identity and 26 % similarity; and CTVL1 vs. CTVL2

showed 17 % identity and 28 % similarity.

The position of the putative cleavage site for each protease differed considerably

between the potyvirus and closterovirus groups, but in all the cases the Gly-Gly pair was

present. A dot matrix generated by Align X of the putative CTVL1 and CTVL2 cysteine

proteases and BYV L-pro is shown in Figure 2. 1B. In this matrix it can be observed that

the more conserved regions are in the areas surrounding the catalytic amino acids, which

are located at the beginning and the end of the plots.








21

The sequence conservation among the two putative cysteine proteases of CTV as

well as their similarity in size have suggested that these proteins evolved in the CTV

genome by a tandem duplication phenomenon (Karasev et al., 1995). Even though gene

duplication with subsequent divergence is a common evolutionary mechanism observed

in DNA genomes, only a few examples are reported for viral RNA genomes (Boyko et

al., 1992). The presence of a duplicated papain-like thiol protease also has been

predicted or identified for the genomes of Coronaviridae and Arteriviridae of the order

Nidovirales (Kanjanahaluethai and Baker, 2000; Zieburhr et al., 2000; Tijms et al., 2001

and references therein).

Gene duplication is also reported for the coat protein gene of the Closteroviridae

family; where a diverged copy of the coat protein (p27) appears in the genome (Boyko et

al., 1992; Febres et al., 1994; Agranovsky et al., 1995). It has been suggested that the

CP duplication occurred in the common ancestor of BYV and CTV (Boyko et al., 1992).

For this duplicated CP the degree of conservation among both proteins was 30 %

similitude (Boyko et al., 1992).

In the present study, the proteolytic activity of the N-terminal region of the CTV

ORF I translation product was demonstrated by in vitro transcription and translation

assay. Expression constructs encoding either the CTVL I and/or CTVL2 were generated

and used in a wheat germ-coupled transcription and translation system. Translation

products were subjected to polyacrylamide gel electrophoresis analysis, and the

proteolytic activity was determined based on the migration patterns of the products

observed in the gels.
















20 40 60
g HD&F;@ j -----------RRADLSURRALG PTVGAFKTYL*EYGR
MK------- R-R!FRIRDVDLGPF PRIWFHRLERLYGK
P A. LC AO ---------- .TFR ............T KL


T2KPI
T2KPII
BYV
GVLRV
PPV
PVY
SMV
TEV
BCMV
TVMV




T2KPI
T2KK::

7 iLRV
PPV
?V
TEV
51CMV
S-IMV


1 ~ ~ r -- -






/Y / /2o
//

I. /
0// "7
* "J/ 1 / ,
7 / // 5
7'' /
'V // '7 /
/, /~ /,
// ", ,'
L -, : ;,z i i ._

BYV L-pro


I, /

1 /


1/


r' - -


/ "' i

/, //

/ '-I

/ .7/,
/ 7


BV- I,

BYV L-pro


,7-
,- *. /
/ /
I, /


/ I
:/ ., ,
/7 /



CTVL2


Figure 2.1: Protein sequence analysis of described and putative cysteine proteases of
potyviruses and closteroviruses. (A) Protein alignment of C-terminal cysteine protease
regions. Closterovirus: T2KPI, citrus tristeza virus CTVL 1; T2KPII, citrus tristeza virus

CTVL2; BYV, beet yellows virus L-pro (Accession Number X73476). Potyvirus:
GVLRV, grapevine leafroll virus (AF039204); PPV, plum pox virus (AJ243957); PVY,
potato virus Y (AF229174); SMV, soybean mosaic virus (S42280); TEV, tobacco etch
virus (NP062908); BCMV, bean common mosaic virus (U19287); TVMV, tobacco vein
mottle virus (X04083); ZYMV, zucchini yellow mosaic virus (L31350). (B) Dot matrix
generated by Align X of the putative CTVL I and CTVL2 cysteine proteases and BYV L-
pro. The conditions to generate the matrices were 30 % stringency with a window size of

5 residues.


80 100 120
DSLKFMR(;T:TFSVF .LStES4-DLRS PNHHLVGG-----------------------
AASRYGVRGYYSAPRCF CYNDSPK-PLTS YHNG G-------------------------
SALKHCVRGRlVSRSLF DVASAFS#PFYS 'FIG -----------------------
RSLC ILYGAYTSR(G DYDAKF]KDLR SAVIAGKDG EVV2SDTPAMXQKTIEAVY
RNARJ LDHEAKIF DIG, ATQ 14NQ ,S#ASD-ThOSSM4TXLvGG--
HDAS I#VDHDTQT. SzGiZTT, SVYSQ#.LAND-ELESIIHYRVGG--
RflARM LVD ACOTM GI04WND SKXYR~
.RAE~LVDHDNKT -R!T TSQLXEVHS-;LESEKXYNVGG;--
RSAR2DUV~iASQM DS GGfNQL.JQNrASl- DLEGENXHYRVGG~-
ASA ILYAKT TT ?jSQL;EKIASN-TLESPNAQYKVGG--











Materials and Methods

Virus Isolate

The Florida grapefruit stem pitting CTV isolate 3800 was used as the RNA

source. This isolate is believed to contain at least two distinct viral strains designated as

T2K and T38K (Manjunath et al., 2000). Sequences of the T2K strain were used in the

present study.

RNA Isolation and Complementary DNA (cDNA) Synthesis


Double stranded (dsRNA) replicative forms of CTV were isolated from bark

tissue of grapefruit plants following the protocol described by Valverde et al., (1990).

The dsRNA was denaturated by incubation at 70 'C for 5 minutes and quickly transferred

to ice. The oligonucleotide 5'-GTCAAACGAGATATCTTTGTCGAGG-3' was used to

specifically prime the Thermoscript (Gibco BRL) mediated reverse transcription of the

T2K sequence.

Polymerase Chain Reaction. Amplification of Protease Domains and Cloning Strategy

Polymerase chain reaction (PCR) was used to amplify different portions of the

coding region for the protease domains of ORF 1. Amplification was performed using

2.5u/100 gl of expand polymerase (Boheringer) in reactions containing 50 mM KCI, 10

mM Tris-HC1 pH 9.0, 0.1% Triton X-100, 2.5 mM MgC12, 0.1 mM of each

deoxyribonucleotide triphosphate (dNTPs), 100 pmol of each primer, and 2 5 ptl of

cDNA. The PCR parameters consisted of 92 "C for 2 min., followed by 30 cycles of 30

sec. at 92"C, 30 seconds at 50*C and 1 minute/Kb at 72C.









24

Figure 2.2 shows a schematic representation of the expression constructs used in

these experiments. Translations of these constructs were expected to contain both

unprocessed products with the full size protein as well as processed products with lower

molecular mass as a result of in vitro proteolytic cleavage.

The primers used to amplify each construct are listed in Table 2.1. Either ApaI

(GGGCCC) or XhoI (CTCGAG) recognition sequences were included within the primer

sequence to facilitate further cloning and manipulation.






G484 G485 G976 G977




CTVL1-L2-PCS2 113 KDa

C VL1-PCS1 59 I I I 1KDa

PCTS-CTVL2PCS2 64 KlDa







Figure 2.2: Schematic representation of CTV ORF I N-terminal region and expression
constructs of CTV papain-like cysteine proteases. The putative cleavage sites that delimit
each protease are denoted by G4"4-G4"5 and G976-G977. Construct CTVL I -PCS I starts with
the first Met of CTV ORFI and ends 40 amino acids after the first putative cleavage site
(PCS). Construct PCS I -CTVL2-PCS2 starts 40 amino acids before the first PCS and
ends 40 amino acids after the second PCS. Construct CTVL I-L2-PCS2 starts at the first
Met of CTV ORF 1 and ends 40 amino acids after the second PCS.
(*) Marks the position of the putative active amino acids C3-H' and C896-H956.











Table 2.1: Sequence of oligonucleotides used to amplify papain-like cysteine protease
domains from ORF I of CTV strain T2K. Restriction sites are shown in bold italic.
Primer: Sequence
CTVL 15' 5'-AAAGGGCCCACCATGTCGAAACTCAGAGGAAGCTT-3'

CTVL1-S1 3' 5'-AAACTCGAGTCAGAGATAACCATCACGTCGCAAGCG-3'

PCS I-CTVL2- 5'-AAAGGGCCCACCATGGACTCTCTTAAGGTTCCTATG-3'
PCS2 5'
PCS 1-CTVL2- 5'-AAACTCGAGTCAGTCCTTTTCCACAGACCGAATC-3'
PCS2 3'
PCSI-CTVL2 3' 5'-AAACTCGAGTCAACCCATATTATGGTACTTATTTAA-3'

CTVL2-PCS2 5' 5'-AAAGGGCCCACCATGGGGACTTCTGCACACGTCTTAAT
TGGGG-3'


Following PCR, the products were GeneClean (Bio 101, Inc.) gel purified and

cloned into the pGEM-T vector (Promega). Selected clones were subjected to restriction

enzyme analysis, and those which were oriented under the control of the SP6 promoter

were submitted for DNA sequencing.

In vitro Transcription and Translation

Selected constructs were sequenced and used as templates for in vitro

transcription and translation reactions. The TNT coupled in vitro transcription and

translation wheat germ extract system (Promega) was used following the manufacturer's

instructions. Briefly, 10 il reactions contained 2 ig of plasmid DNA/pil and were

incubated at 30'C for 1 hour in the presence of 0.5 IiCi/ml [3H] leucine. After

incubation, the reactions were subjected to sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) in 7.5 % gels for one hour at 200V. For detection of the

radiolabeled proteins, the method described by Bonner and Laskey (1974) was followed.











Briefly, the gels were fixed in dimethyl sulfoxide (DMSO) and DMSO- 2,5-

diphenyloxazole (PPO). Following hydration-PPO precipitation, the gels were dried and

exposed to x-ray film overnight at -80'C, and developed using an automatic X-ray film

developer (Kodak X-Omat Clinic 1 Processor).

Protease Inhibitor Assays

The sensitivity of the proteolytic reactions was tested by the addition of different

chemical protease inhibitors to the translation mixture. In vitro transcription and

translation reactions were performed as described above with the addition of one of the

following cysteine protease inhibitors: 100 [.M trans-epoxysuccinil-L-leucylamido-(4-

guanidino) butane (E-64); 2.5 mM N-ethylmaleimide (NEM); or a broad spectrum

protease inhibitor cocktail containing 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride

(AEBSF), 0.8 .LM aproptinin, 20 pM leupeptin, 36 p[M bestatin, 15 pM pepstatin A and

14 pM E-64.

The translation mixtures were incubated at 30 'C for 1 hour and the products

analyzed by SDS-PAGE.

Results

CTVL I is Proteolytically Active

Analyses of the proteolytic activity of the two putative cysteine proteases of CTV

ORF I were performed by the construction of three overlapping cDNA constructs that

spanned 640 amino acids from the N-terminal region of ORFI to include the two PCPs

and the predicted cleavage sites. These cDNAs were cloned into the pGEM-T vector

under the control of SP6 polymerase, subjected to coupled in vitro transcription and











translation, and the resulting products were analyzed by SDS-PAGE and

autoradiography. The first construct, CTVL I-PCS 1 (Figure 2.2), started at methionine 1

of CTV ORFI and ended 40 amino acids downstream of the first PCS. The expected

product from this translation was a full length protein with a mass of approximately 58

KDa. If this protein had autoproteolytic activity, it was expected to liberate the 40

downstream amino acids of the PCS G8-G4, producing an additional product of 54

KDa, corresponding to the mature proteolytic product. The SDS-PAGE autoradiogram

of the CTVL 1 -PCS 1 translation product is shown in Figure 3B. Two proteins are present

which correspond to the predicted full size protein (58 KDa) and the mature product of

CTVL 1-PCS 1 (54 KDa). This result confirmed the proteolytic activity of the first PLP

domain of CTV ORF 1.

No Proteolytic Activity was Detected for the CTVL2 Protein

To test the proteolytic activity of the second PLP of CTV ORF 1, the construct

PCS1-CTVL2-PCS2 was designed (Figure 2.2). To design this construct, we had to take

into consideration that CTVL2 is an internal domain of the polyprotein, and that it may

be released from it by proteolysis. This raised three main concerns for the design of this

construct. First was the possible effect on the activity of the protein by the introduction

of an extra amino acid in order to have methionine as the first amino acid for its

expression; second was that the second PLP domain was delimited by a PCS at its N and

C terminal ends, and both PCSs had to be considered as potential substrates; and third

was the length of the amino acid chains that were either before or after the PCS,

designated as the substrates.











These substrates were designed to determine proteolytic activity for each PLP,

based on size differences as determined by the difference in electrophoretic mobilities

between the unprocessed and mature proteins. To detect proteolytic activity, the mass of

the substrate needed to be detectably different when compared to the size of the protein

carrying the proteolytic activity. Due to the C-terminal proximity of the PLP on each

protein, the substrate peptide also needed to be small enough so that it would not include

the first PCP active site in the construct for the second PCP (see Figure 2.2). Therefore, a

length of 40 amino acids was chosen to exclude the first catalytic domain from the

substrate of PCS1-CTVL2-PCS2. To be consistent with this length limitation, this same

length of amino acids was used in the design of the rest of the constructs.

Translation of PCS 1 -CTVL2-PCS2 was expected to produce a full length product

of approximately 64 KDa. Proteolytic processing at both PCSs was expected to produce a

54 KDa protein. Also, it was anticipated that an intermediate protein of 58 KDa

corresponding to partially processed substrates at either PCS might be present.

The SDS-PAGE autoradiogram of PCS 1 -CTVL2-PCS2 translation products is

shown in Figure 2.3C, lane 1. Only one product of approximately 64 KDa was obtained

for this construct. The estimated molecular mass for this protein corresponded to the

predicted full size for PCS 1-CTVL2-PCS2. This result seemed to indicate that there was

no proteolytic activity associated with CTVL2 at either of the PCSs. To rule out the

requirement of a cleavage at the first PCS in order to activate the second PLP domain, the

construct CTVL2-PCS2 (Figure 2.3 C, lane 2) was translated.














A 44 G45 G976 0977 kDa
Molecuar mass 216

113 KDa CTVL1-L2-PCS2 132
59 KDa CTVLI-PCS I 78
54 KDa CTVL1
64 KDa PCSI-CTVL2-PCS2 52
59 KD& CGVL2-PCS2 'A 46
59 KDa PCSI CTVL2 32
54 gDa CTVL2


Figure 2.3: Analysis of proteolytic activity of the N-terminal region of CTV ORFI. (A)
Schematic representation of the expected proteins from the translation constructs used for
in vitro transcription and translation and the possible mature products, intermediates and
expected molecular masses. (B-D) SDS-PAGE autoradiograms of [3H] Leu labeled wheat
germ coupled in vitro transcription and translation products of CTV PCP constructs. (B)
CTVL1. (C) PCSI-CTVL2-PCS2, CTVL2-PCS2 (D) CTVL1-L2-PCS2.


Construct CTVL2-PCS2 represented the beginning of the second PCP, starting

with an additional methionine followed by the glycine that occupies the P1' position at

the first PCS. It also included the second PCS. This construct was intended to simulate

the protein after being cleaved at PCS 1 by CTVL 1. Translation of construct CTVL2-

PCS2 produced a full sized protein of 58 KDa, but not a mature 54 KDa product (Figure

2.3C, lane 2). Again, this second construct, which was designed to demonstrate

proteolytic activity in the CTVL2 protein failed to demonstrate this activity. Even

though proteolytic activity from CTVL2 was not detected, it can not be assumed that this

protein is proteolytically inactive.









30

Processing Pattern of the N-terminal Region of CTV ORF I Revealed Three Cleavages in
the Region

To study the proteolytic activity of the whole N-terminal region of CTV ORF 1 as

a unit, we translated the construct CTVLl-L2-PCS2. This plasmid encodes both

proteolytic domains as well as both cleavage sites. The predicted products from this

experiment are the full size protein of an estimated molecular mass of 113 KDa and, after

full processing at the PCSs, two proteins of undistinguishable molecular mass of

approximately 54 KDa corresponding to CTVL 1 and CTVL2. Also, depending on the

maturation pattern that this region follows, processing intermediates of CTVL2-PCS2

can be expected to be released after a first cleavage at PCS 1 or of CTVL I -L2 after a first

cleavage at PCS2 (see Figure 2.3A).

The translation products of CTVL 1 -L2-PCS2 are shown in Figure 2.3D. The

banding pattern revealed four distinctive proteins with approximate molecular masses of

120, 60, 57 and 54 KDa. These proteins can be assigned to the full sized unprocessed

protein (120 KDa), proteolytic intermediate CTVL2-PCS2 (60 KDa) and fully processed

CTVL 1 and CTVL2 (57 KDa). The fourth protein of 52 KDa that appears in the gel is the

predominant product in the translation mixture, and its size can not be related to any of

the predicted proteolytic products.

Sensitivity to Protease Inhibitors

Protease inhibitors have been used in the characterization of the enzymatic

activity of proteases. To test the sensitivity ofCTVL I and CTVL2 to chemical

inhibitors, we performed in vitro transcription and translation assays in the presence of

cysteine protease specific inhibitors and a broad spectrum protease inhibitor cocktail.











As shown in Figure 2.4, the presence of the cysteine protease inhibitors E-64

(Figure 2.4) or NEM (data not shown) did not affect any of the proteolytic reactions at

the concentrations tested. No effect of the protease inhibitor cocktail was observed as

well. The decrease in transcription-translation efficiency in the presence of protease

inhibitors reflects their interference with the translation system.


N
Q
N


COCKTAIL


/


E64


Figure 2.4: Effect of chemical protease inhibitors on the proteolytic activity of CTVL 1
and CTVL2. In vitro transcription and translation of CTVL 1 -PCS 1 and CTVL 1 -L2-
PCS2 in the presence of a broad spectrum protease inhibitor cocktail (1 mM AEBSF,
0.8ViM aproptinin, 20 V.M leupeptin, 36 [iM bestatin, 15 VM pepstatin A and 14 vM E-64)
or the cysteine protease specific inhibitor E-64 (100 pM). The presence of the protease
inhibitors did not change the processing pattern of these constructs, but it did affect the
transcription-translation efficiency of the system.











Discussion

Sequence analysis has revealed the presence of PLPs in different members of the

family Closteroviridae (Agranovsky et al., 1994; Karasev et al., 1995; Klaassen et.. al.,

1995; Jelkmann et al., 1997 and Zhu et al., 1998). For all PLPs reported for

Closteroviridae, L-pro from BYV is the only case for which the PCP has been

experimentally tested and its proteolytic release from the polyprotein demonstrated in an

in vitro transcription and translation assay (Agranovsky et al., 1995).

The data presented here corroborates the catalytic activity of the N terminal

region of CTV ORF1. Fully processed products of CTVLI-L2-PCS2 showed at least

four major processed proteins, accounting for three cleavages within this region. There

were also several minor proteins that were present to some extent in all the translation

reactions. The origin of these minor proteins has not been determined. N-terminal

sequencing of those products could help to elucidate if they are produced by proteolysis

or not. Alternatively, these minor proteins may not be proteolytic products but rather be

artifacts of the system where early termination and internal initiation is known to occur.

The same concern was reported during in vitro translation assays of the equine arteritis

virus papain protease (Snijder et al., 1992), where similar minor proteins were

disregarded as artifacts of the system since these proteins also occurred in the presence of

protease inhibitors. In our case, the presence and quantity of these bands also varied

accordingly to the age of the DNA used in the translation reaction (data not shown).

The proteolytic activity for CTVL 1 was demonstrated by in vitro transcription

and translation. For this protein a mature product appeared to be produced by proteolysis











at the predicted cleavage site G4-G"'5, based on the expected molecular masses for the

products present in the reaction. Based on these results, autoproteolytic release of

CTVL I from the polyprotein is comparable to that reported for BYV L-pro (Agranovsky

et al., 1994). So far we have expressed CTVL2 in the presence of a PCS at both N- and

C- termini, and we removed the N-terminal PCS to simulate the cleavage of PCS I by

CTVL 1. In neither case was proteolytic activity observed. However, the lack of activity

of CTVL2 in our system does not necessarily rule out catalytic capacity for this protein.

The first explanation could be that CTVL2 might not be functional at either of the

predicted cleavage sites. Alternatively, other factors might be necessary to modulate the

activity of this protein. It has been shown that activity of PCPs can be greatly influenced

by the length of the substrates and of the protein itself (Teng et al., 1999).

There is a similar report of inactivity of a second PLP in the murine coronavirus

mouse hepatitis virus (MHV) ORF 1. In this case, the lack of activity of the MHV PLP-2

protease was due to the experimental design which was testing for activity on a substrate

described for the other protease in the viral genome. It was not until the substrate for this

protein was identified hundreds of amino acids downstream of the PLP2 domain (Schiller

et al., 1998) that the activity of this protein was finally characterized (Kanjanahaluethai

and Baker, 2000). A similar explanation may pertain for CTVL2 in our experiments.

The substrates presented to this protease were only the predicted cleavage sites G484-G485

and G976-G977. An alternative possibility could be that the substrate for this enzyme is not

any of the PCSs predicted by Karasev et al. (1995) but an unreported site elsewhere in

the polyprotein, or possibly in any other protein of viral or cellular origin.









34

The translation of CTVL 1 -L2-PCS2 evidenced at least three cleavages within the

area expressed. From these data, we can not determine if all three cleavages were

mediated by CTVL 1, or if there was participation of CTVL2 in the maturation process.

The molecular mass (52 KDa) of the principal product in the translation reaction

did not correspond to the products predicted after cleavage at both PCSs. Thus the

appearance of this protein may result from cleavage at a previously unreported site. The

presence of a duplicated PCP has been reported for other (+) ssRNA viruses besides

closteroviruses (Lee et al., 1991; Shapira and Nuss, 1991; Godeney et al., 1993).

Although it seems to be a common phenomenon, there is not yet an explanation of a

selective advantage for this duplication (Garcia et al., 1999). Another common finding is

the presence of more than one catalytic type of protease encoded in the same viral

genome, performing sequential cleavages based on different affinities for different

substrates that might be modulated by accessibility and local conformation (Bergmann &

James, 1999; de Groot et al., 1990). The present study was only on the proteolytic

activity within the N-terminal region of CTV ORF 1. There is evidence that the

polyprotein encoded by closterovirus ORF I is processed in a pattern similar to that of the

precursor of nonstructural proteins of alphaviruses and I a/ lb polyprotein of corona-like

viruses (Erokhina et al., 2000). The presence of methyl transferase and helicase domains

as single proteins released from the polyprotein was demonstrated by the use of

monoclonal antibodies in BYV infected tissue. This indicated that closterovirus ORFI

protein undergoes multiple proteolysis in vivo (Erokhina et al., 2000). The RNA

dependant RNA polymerase of CTV has also been demonstrated to be released from the









35

polyprotein. Using antibodies, Cevik (personal communication), was able to locate CTV

RdRp in cell fractions of CTV-infected tissue. The nature of the proteins that mediate the

maturation of the C-terminal region of closterovirus ORF l a/b have not been identified,

but it has been suggested that this processing might be mediated by a trans activity of L-

pro (or L-pro-like) or by a host enzyme (Erokhina et al., 2000). The results obtained with

the protease inhibitor study revealed that both CTVL 1 and CTVL2 were insensitive to

the inhibitors tested under the conditions present in our system. It has been reported that

protease inhibitors are capable of blocking the translation reaction (Pieroni et al., 1997),

and several investigations took advantage of post-translational processing or trans

proteolytic activity assays to perform these studies (Hahm et al., 1995; Pieroni et al.,

1997; Sircar et al., 1998; Hata et al., 2000). For CTVLI and CTVL2, it seems to be a co-

translational process, and even though we observed decreased concentration of the

translation products in the presence of protease inhibitors, we were able to observe

mature translation products in assays with two different concentrations of the E-64

inhibitor. The insensitivity of some viral cysteine proteases to the E-64 inhibitor has

been reported previously (Tihanyi et al., 1993; Sircar et. al., 1998; Andr6s et al., 2001).

A study of the activity of the African swine fever virus protease revealed its insensitivity

to E-64, but it was susceptible to the cysteine protease inhibitor NEM (Andr6s et al.,

2001). In the case of CTVL 1 and CTVL2, both were insensitive to E-64 and NEM or to

any of the protease inhibitors present in the cocktail used in our experiment. This

insensitivity to protease inhibitors may add to the characteristic properties of these viral

proteases.














CHAPTER 3
SITE DIRECTED MUTAGENESIS OF PUTATIVE ACTIVE AND CLEAVAGE SITES
OF CTVL 1 AND CTVL2: THE N-TERMINAL PROCESSING DYNAMICS.

Catalysis by the cysteine proteases proceeds via the formation of a covalent

intermediate and involves a cysteine and a histidine residue (Polgar and Halasz, 1982).

Usually, for papain-like cysteine proteases, the catalytic cysteine is flanked at the C-

terminal site by tryptophan, a bulky hydrophobic amino acid (Gorbalenya et al., 1991).

A comparison between cellular and viral cysteine proteases showed that the

spacing between the two catalytic residues differs considerably. For cellular papain-like

proteases, the space between these amino acids is approximately 130 to 160 residues,

contrasting with the viral proteases having approximately 60 80 residues (Snijder et al.,

1992). These characteristics and other properties derived from sequence alignments of

cellular and viral proteases have allowed the identification of new putative papain-related

thiol proteases encoded by the genomes of the positive-strand RNA viruses (Gorbalenya

et al., 1991).

In the identification of the closterovirus BYV L-pro cysteine protease, sequence

alignments revealed the JxxxxGOCYU motif (J= aromatic, X= any amino acid, 0= bulky

aliphatic or aromatic, U= bulky) to be the consensus sequence for the closterovirus and

potyvirus families (Agranovsky et al., 1994). The consensus sequence BGxCYUxH was

reported for the Closteroviridae family (Karasev et al., 1995). From this analysis, the

most conserved region around the catalytic cysteine was the dipeptide CW(Y), being









37

characteristic of these kinds of proteases (Gorbalenya et al., 1991). The other amino acid

in the catalytic dyad is histidine, and the sequence conservation around it is lower than

that found around the catalytic cysteine, making predictions for this amino acid position

more difficult (Gorbalenya et al., 199 1; Agranovsky et al., 1994).

Protein alignments for closterovirus sequences had identified the presence of a

PCP domain in the N-terminal region of BYV ORF 1 translation product, identified by its

similarity to those PLP from potyviruses. Site directed mutagenesis of C509 and H596

completely abolished proteolytic activity of BYV-L-pro demonstrating the involvement

of these residues in the proteolytic reaction (Agranovsky et al., 1994). Sequence analysis

of the CTV genome revealed two PCP domains encoded by ORF I that represent a gene

duplication when compared with the genome of BYV (Karasev et al., 1995). From the

sequence alignments with BYV L-pro, C4"3 H4" were predicted to be the active amino

acids for the first proteolytic domain and C896 H956 for the second domain in the CTV

ORF1.

In the description of the interaction between a protease and its substrate, the

conventional nomenclature refers to the protease subsites as "S" and the substrate amino

acids as "P". The amino acids of the N-terminal side of the scissile bond are numbered

P3, P2, P1 and those of the C-terminal side are numbered PI', P2', P3'..., where the bond

between PI-Pl' is the scissile bond. Similarly, the protease subsites that complement the

substrate binding site are numbered S3, S2, S1, S l', S2', S3' (Schechter and Berger, 1967).









38

Generally, leader proteases have been reported to cleave between two small amino

acid residues (Carrington et al., 1989; Gorbalenya et al., 1991; Kirchweger et al., 1994).

With the exception of the conserved dipeptide CW(Y), alignment of viral cysteine

proteases revealed little conservation (Gorbalenya et al., 1991). The amino acid

conservation around the putative cleavage sites of closterovirus cysteine proteases is

limited to the P2, P 1 and P4' positions, where a bulky hydrophobic residue, a Gly residue

and a negatively charged residue are usually found (Jelkmann et al., 1997; Zhu et al.,

1998).

Studies on the putative substrate for BYV Lpro, revealed that mutations at the

predicted P 1 position G588 abolished the proteolytic processing of the N-terminal region

of BYV (Agranovsky et al., 1994). By comparison with the BYV sequence, two putative

cleavage sites were predicted at G484-G485 and G976-G 977 for the CTV ORF I protein

(Karasev et al., 1995).

In this study, site directed mutagenesis was performed to both putative active site

amino acids for each proteolytic domain, as well as for the amino acid that occupies the

P1 position at each putative cleavage site. These constructs were used in an in vitro

transcription and translation assay to determine the effects of the mutations on the

proteolytic processing observed for the N-terminal region of CTV ORF 1.

Materials and Methods

The CTV strain used and the general RNA reverse transcription and DNA

amplification techniques were as described in Chapter 2.











Site Directed Mutagenesis

Site directed mutagenesis (SDM) was performed using a PCR amplification

technique. Two partially overlapping complementary oligonucleotides carrying the

desired mutation were used to prime individual amplifications with the corresponding 5'

or 3' oligonucleotide pair. Overlapping PCR mutated products were mixed and

reamplified using the external primers to generate the complete coding sequence for the

desired protein. The primers used to introduce the mutations in the putative active sites

are shown in Table 3.1.

Table 3.1: Oligonucleotide primer sequences used to introduce mutations to the putative
active sites. The mutagenized codons are shown in bold italic letters.
Primer Sequence

SDM C403S+ 5'-CGGTCAGA GC'ATGTCCGTCACGTGTTC-3'

SDM C403S- 5'-CGGACATAGCTCTGACCGTCGCGAACTTTAGC-3'

SDM C896S+ 5'-ATCCCTGAAGGAAGGATATAGCTACATTCGC-3'

SDM C896S- 5'-GCGAATGTAGCTATATCCTTCCTTCAGGGAT-3'

SDM H646E+ 5'-GGTAGTGTTTTTGAATGCTTGTCA-3'

SDM H646E- 5'-TGACAAGCATTCAAAAACACTACC -3'

SDM H956E+ 5'-CCACGCTGCTTCGAATGTTGCTAC-3'

SDM H956E- 5'-GTAGCAACATTCGAAGCAGCGTGG-3

Several mutations were introduced in the P 1 position of both PCSs. SDM was

performed using a common antisense oligonucleotide that did not carry any mutation and

had an overlap with conserved regions of the mutagenic sense primers. These primers are

shown in Table 3.2.











Table 3.2: Oligonucleotide primer sequences used to introduce mutations into the
putative cleavage sites.


Primer


Sequence


PCS1 -

SDM G484A+

SDM G484M+

SDM G484E+

SDM G484F+

PCS2 -

SDM G976S+

SDM G967A+

SDM G967M+

SDM G976E+

SDM G976F+


5'-AACTAAATGATGGTTAGGAATAGA-3'

5'-AACCATCATTTAGTTGCCGGGACTTCT-3'

5 '-AACCATCATTTAGTTA TGGGGACTTCT-3'

5'-AACCATCATTTAGTTGA GGGGACTTCT-3'

5'-AACCATCATTTAGTTTTCGGGACTTCT-3'

5'-CATATTATGATACTTATTTAA-3'

5'-TATCATAATATGA GCGGCGAAGAT-3'

5'-TATCATAATATGGCCGGCGAAGAT-3'

5'-TATCATAATATGA TGGGCGAAGAT-3'

5'-TATCATAATATGGAGGGCGAAGAT-3'

5'-TATCATAATATGTTCGGCGAAGAT-3'


In vitro Transcription and Translation


Mutagenized proteins were used in an in vitro transcription and translation assay

as described in Chapter 2.

Results

Cysteine and Histidine are Involved in CTVL 1 Activity

Using an in vitro transcription and translation assay, the autoproteolytic activity of

CTVL 1 over the PCS 1 in the CTVL 1 -PCS I construct was demonstrated (Chapter 2).

This construct produced two proteins one "precursor" with molecular mass of


approximately 59 KDa and a mature protein of 54 KDa (Figure 3. 1B).











To identify the amino acids involved in the proteolytic activity of CTVL 1, we

performed SDM of the predicted catalytic residues C'3 and H', producing constructs

carrying individual mutations at either amino acid. Translation from CTVLl c403A-PCS 1

(Figure 3.1B) as well as CTVLl H403E-PCS1 (data not shown) produced a unique band with

a molecular mass corresponding to that of the precursor or unprocessed protein. Even

though we can not unequivocally conclude that C43 and H4' are the catalytic amino

acids, the loss of activity in the mutants suggests their relevance with respect to the

catalytic activity of CTVL 1.

CTVL I Might be Responsible for More Than One Cleavage Within the CTV ORF I N-
Terminal Region

After demonstrating that mutations at either C"3 or H41 inactivated CTVL 1

proteolytic activity, the same mutations were introduced into the CTVL 1-L2-PCS2

construct to study the overall effect of CTVL 1 on the processing of the CTV ORF I N-

terminal region.

It was demonstrated in Chapter 2 that there were at least three proteolytic

cleavages within the region, producing four distinct products (Figure 3. 1B). Mutations at

either active amino acid of CTVL 1 completely changed the processing pattern observed

for the wild type protein. These constructs produced a pattern of one doublet with

molecular mass of approximately 120 KDa that corresponded to the predicted mass of the

unprocessed protein encoded in these constructs and the intermediate molecule after

PCS2 cleavage. These results demonstrated the activity of CTVL2 on PCS2, releasing it

from the polyprotein, and also showing that CTVL 1 might have a more extensive role in

the proteolytic processing of the CTV ORF I N-terminal region. An alternative








42

explanation is that CTVL I might initiate a proteolytic activating cascade, and its activity

is required to activate downstream proteolytic events mediated by CTVL2.

CTVL2 is an Active Protease

Previously we were unable to demonstrate any proteolytic activity for CTVL2

when studied with the PCS 1-CTVL2-PCS2 and CTVL2-PCS2 constructs (Chapter 2). To

determine whether CTVL2 has an active role in the proteolytic processing observed in the

CTVL 1 -L2-PCS2 construct, we introduced mutations at each of the two CTVL2 putative

catalytic amino acids in individual clones. As mentioned above, CTVL2 was shown to

perform the proteolytic processing of PCS2, evidenced when CTVL 1 was inactivated. To

determine if the predicted amino acids were the catalytic residues, we performed SDM of

C896 and H956 These substitutions when introduced changed the processing pattern

observed for the translation products when compared to the CTVL1-L2-PCS2 wild type

(Figure 3.1, lanes 4 and 5). Both mutations produced the same pattern, and it differed

from the wild type protein with respect to the absence of the 53KDa protein. This protein

corresponds with the extra protein that was not predicted for the translation of CTVL 1-

L2-PCS based on the presence of two putative cleavage sites. This suggests that CTVL2

is an active protease, which cleaves at both PCS2 and at an unpredicted site. The putative

active site amino acids for CTVL2, C896 and H956, seem to have a key role in the

proteolytic activity of the enzyme.

CTVL I Might be an Additional Substrate for CTVL2

A second activity of CTVL2 was demonstrated by an additional change in the

processing pattern of CTVL 1 -L2-PCS2 when CTVL2 putative active amino acids were









43

mutated. The substrate for CTVL2 might be within the area of CTVL 1 since its activity

was not detected when it was tested on the PCS 1-CTVL2-PCS2 construct and was

evident only when both proteases were expressed together.

To study the characteristics of the putative cleavage sites, we introduced the

mutation G484S in the P1 position of PCS1 in the CTVLI-PCSI construct. Translation

from this mutant produced a processing pattern similar to the one observed for CTVL I-

PCS1, having an unprocessed protein of 59 KDa and a mature protein of 54 KDa. In

addition to these two proteins, a third protein of 53KDa appeared for the CTVL 1-PCS484S

translation product (Figure 3.2, lane 2). This additional protein co-migrated with the

unpredicted 53KDa band in the CTVL1-L2-PCS2, seemingly present as a product of the

activity of CTVL2. If these two co-migrating bands are the same protein, this result

indicates that a cleavage site for CTVL2 is within the area of CTVL 1. Even though

CTVLI does not normally cleave at this site, it is not surprising to see proteolytic activity

at an alternative cleavage site. When the wild type substrate is modified, especially if

both proteolytic activities are derived from a gene duplication event, they might still have

overlapping activities as they diverge from each other


















Af


G"G485 G9176 Gg77



Molecula=r =a

59-5 kDa CTVLI-PCS!



59 kDa CTVLlm-PCSI


113 kDa CTVLI L2-PCS2
C403S/A



I113 kDa CTVLI. L2LPCS2
403SIA
113 ~ ~ ~ ~ k: 7L-~iPS


IN


Y -





U


-216


-132

- 78


- '- 59
-54


,Je"


- 46


- 32


Figure 3.1: Site directed mutagenesis of predicted catalytic amino acids of CTVL1 and

CTVL2. (A) Schematic representation of the constructs used in this experiment.

Constructs carrying individual mutations were C403S, C403A, H646E, C896S, C896A,

H956E. (B) SDS-PAGE autoradiograms of [3H]Leu labeled wheat germ coupled in vitro

transcription and translation products of mutagenized CTVL I-L2-PCS2 constructs.

Mutations shown correspond to C403S and C896S.


kDa


6


G464 C485 G976 G977

Molecular inass


59 kDa CTVLI-PCSI



59 kDa CTVL I-PCSIl,,,





C986S

113 kDa CVL I-L2,-PCS2


78



59
54
52


46


32


Figure 3.2: Site directed mutagenesis of PCS 1. Localization of CTVL2 substrate site. (A)

Schematic representation of the constructs used for in vitro transcription and translation.

(B) SDS-PAGE Autoradiograms of [3H] Leu labeled wheat germ coupled in vitro

transcription and translation products.










Mutations at the P1 Position of PCS1 and PCS2 are not Well Tolerated

Mutations at either PCS produced similar phenotypes to those observed when the

active amino acids were mutated, but they did not completely abolish processing in the

majority of the cases (Figure 3.3). Glycine at the P1 position of each PCS was substituted

by an alanine, serine, methionine, phenylalanine and aspartic acid on the CTVL 1 -L2-

PCS2 construct. Mutation G484A at PCS1 reduced the processing of the whole product,

causing an increase in the unprocessed full size protein when compared to wild type

(Figure 3.3, lane 1). Curiously, an important reduction in the activity of CTVL2 was also

observed. A more drastic effect on the proteolytic processing was caused by the less

conservative mutations G484F (Figure 3.3, lane 2), G484M and, G484E (data not shown).

In these cases, the effect of the mutation resembled the CTVL I active site mutations, as

the processing of the protein was drastically reduced for both CTVL 1 and CTVL2

activities.Mutations of PCS2 had the pattern characteristic of CTVL2 active site mutants,

carrying drastically reduced CTVL2 activity. When compared to wild type, the 59 KDa

band that corresponds to CTVL2-PCS2 is in greater abundance in the mutant, evidencing

the reduced processing at the PCS2 (Figure 3.3, lanes 3-6). These constructs also showed

a reduced CTVL2 activity, suggesting that an efficient cleavage at the PCS2 is required

for activation of CTVL2. Mutations G976M and G976E abolished almost all CTVL2

activity (Figure 3.3, lanes 3 and 4), and G976F (Figure 3.3, lane 5), G9976A and G976S

(data not shown) reduced CTVL2 activity but some 53 KDa product was still evident.










1 2 3 4 5 6 7 kDa

-216


132


78




S46

32

Figure 3.3: SDS-PAGE autoradiogram of CTVL 1-L2-PCS2 proteins mutagenized at the
P 1 position of their putative cleavage sties. Lanes I and 2 are mutations of PCS 1. Lanes
3 through 5 are mutations of PCS2. The following lanes contain results with the
indicated mutations: 1) G484A; 2) G484F; 3) G976M; 4) G976E; 5) G976F; 6) CTVL 1-
L2-PCS I Wild type; 7) CTVL 1-L2896A-PCS2.


Discussion

Identification of the residues involved in the catalytic activity of the active

residues of BYV L-pro by SDM (Agranovsky et al., 1994) facilitated the prediction of

putative catalytic amino acids for both putative papain-like cysteine proteases of CTV.

This identification was accomplished based upon sequence alignments of ORF I a of both

CTV and BYV (Karasev et al., 1995). Even though the sequence conservation was

limited between these proteases, the catalytic dyads appeared to be Cys3 and His' for

CTVL 1 and Cys89 and His956 for CTVL2. Protein alignments also enabled the prediction

of cleavage sites for both proteolytic domains. However, there was no experimental

evidence for the identity of the active amino acids for both CTV PCP domains.










In this study we introduced mutations for each of the putative catalytic amino

acids for either PCPD in the CTV ORF I N-terminal region. To identify the catalytic

cysteines, we introduced the conservative mutations C403S and C896S. The other

catalytic position was represented by the residues H' and H956 for CTVL 1 and CTVL2,

respectively. These amino acids were substituted with aspartic acid, which is the most

common substitution of non-conserved histidines in the closterovirus genomes

(Agranovsky et al., 1994).

All the substitutions of the putative catalytic amino acids in each protease changed

the processing pattern observed for the wild type protein. This indicates the importance

of each of these amino acids in the proteolytic processing of CTV ORFI N-terminal

region Even though the identity of these amino acids as the catalytic residues cannot be

unequivocally assured, there are several characteristics that support this conclusion.

Sequence alignment with previously identified catalytic amino acids of the

"closely related" BYV-LPro protease and the change in the processing pattern when

these residues were substituted constitute the strongest line of evidence. There are other

characteristics that are shared with several viral cysteine proteases. These characteristics

include an overall size less than 155 amino acids versus the over 200 amino acids for

cellular proteases. The distance between catalytic amino acids is smaller for viral than for

cellular proteases, and the relative placement of the catalytic amino acids is towards the

C-terminal region of the proteolytic domain (Oh and Carrington, 1989; Gorbalenya et al.,

1991). The demonstration of proteolytic activity in the CTV ORF I N-terminal region

confirms the prediction of the presence of at least one proteolytic enzyme encoded by










CTV. Other members of the Closteroviridae family also were predicted to carry PCP

within their genomes. Sequence alignment for little cherry virus (LChV), lettuce

infectious virus (LIV) and sweet potato sunken vein virus (SPSVV) leader proteins did

not reveal sequence conservation apart from the C-terminal PCP domain, even though

there was stronger conservation among PCP of these Closterovirus or Closterovirus-like

genomes when compared to those of BYV and CTV. Another conserved domain also

was found upstream of the PCP for these three viruses. These data agree with the

grouping derived from the RNA polymerase alignments, suggesting that these two

proteins evolved as a single entity (Jelkman et al., 1997).

When CTVL 1 was mutated in the CTVL 1-L2-PCS2 construct, all major cleavages

were absent, leaving only the processing at the PCS2. This result suggests that CTVL2

has proteolytic activity over the PCS2, being revealed only when CTVL 1 was inactivated.

Previously, we did not find any obvious activity of CTVL2 when it was expressed as an

individual protein out of its polyprotein context. A similar case of a duplicated cysteine

protease is found in the genome of the equine arteritis virus (EAV). For this virus,

expression of single protease domains out of the polyprotein context drastically reduced

their activity, evidencing the importance of the conformation adopted when expressed as

part of a larger protein (den Boon et al., 1995). A similar situation was found for the

murine coronavirus MHV, where the PLP- 1 activity seems to be optimal when both

enzyme and substrate are expressed as a unit or as part of a large protein. Protein

sequences at both the N- and the C-terminal positions of the proteolytic domain affected

its catalytic efficiency (Teng et al., 1999). With CTV we may have a similar situation








49

when CTVL 1 -PCS I is expressed. The catalytic reaction proceeds, but further incubation

of the product after stopping the translation reaction does not result in all the precursor

being processed into mature protein (data not shown), evidencing the inefficiency of the

process. Similarly PCS1-CTVL2-PCS2 did not show any activity when expressed by

itself, but it revealed proteolytic activity when expressed as part of a larger protein.

From these results we can infer a processing pattern in which CTVL 1 and CTVL2

cleave themselves from the polyprotein at PCS 1 and PCS2, respectively. Cleavage at

PCS 1 activates CTVL2 to perform a second cleavage within the area of CTVL 1. Thus in

this system, CTVL 1 co-translationally cleaves itself from the polyprotein and activates a

downstream event. CTVL2 catalyzes at least two proteolytic cleavages; one at its own C-

terminal end on the PCS2 in a CTVL 1 independent fashion and a second cleavage at the

N-terminal region of CTVL 1 and in a CTVL 1 dependent fashion.

Duplicated cysteine proteases have been reported in other viral groups (Lee et al.,

1991; Shapira and Nuss, 1991; Godeney et al. 1993). Arterivirus replicase ORFIa

protein encodes two cysteine protease domains that are located in similar positions to

those of CTV within the viral polyprotein. These PCPDs are designated as nsp 1 c and

nsp 1[3. In this system, nsp Ia releases itself from the polyprotein cleaving the

nsp 1 a/nsp I 3 bond. Nsp I 3 releases itself from the rest of the polyprotein cleaving the

nsp I P/polyprotein bond. The individual cleavages of these two proteins are independent

of each other (den Boon et al., 1995). These results correlate with the autoproteolytic

release of CTVL I and CTVL2 from the polyprotein independent of each other.











A well documented case of cysteine protease duplication is that of murine

coronaviruses. When aligned with that of other coronaviruses and cellular papain

proteases, the sequence of murine hepatitis virus (MHV) revealed the presence of two

putative papain-like protease domains in the viral genome (Lee et al., 1991). When

comparing the position of the active amino acids of the MHV proteases with those of

CTV, we found that the positions of the catalytic amino acids for MHV Prol were C..2'

and H 27, with 157 residues between them, in contrast to the 61 amino acids for CTVL 1.

Similarly, MHVPro2 has 157 residues between its catalytic amino acids while CTVL2

has only 60. Even though there are differences between the lengths of the spacers for

these proteases when compared between the viral groups, the difference is conserved

within the same viral genome, thus being characteristic for the entire duplicated gene.

The separation between the catalytic centers of both PCP is also greater for MHV; 595

residues between the catalytic cysteines compared to the 493 residues for CTV.

One proteolytic activity was reported with the translation of murine coronavirus

gene 1 polyprotein. This protein underwent proteolytic processing releasing a protein of

28 KDa, and that activity was linked to the Cys"3 residue. This activity was detected

only when the whole polyprotein was expressed, revealing the importance of the overall

conformation (Baker et al., 1990). Extensive site directed mutagenesis of the putative

p28 cleavage site and surrounding amino acids revealed the importance of the P1, P2 and

P5 positions in the maturation of this protein. The cleavage site was mapped to the G247-

Val1248 dipeptide, with Gly247 and Arg246 also being major determinants for the recognition

of this cleavage site (Dong and Baker, 1994; Hughes et al., 1995; Dong et al., 1995).










Deletion analysis studies of the p28 processing showed that processing at this cleavage

site was affected by sequences upstream to the PCPD, down regulating its activity. In

addition, some of the deletion constructs revealed a different processing pattern that

coincided with a 65 KDa protein (p65) that was present in virus-infected cells. These

observations suggested the presence of another cleavage site in the region. The catalytic

His1272 was identified, and the activity of the proteolytic domain was demonstrated on

both cleavage sites (Bonilla et al., 1995). The second substrate for the protease, which

released the p65 protein, was characterized and compared to the p28 cleavage site. Both

sequences had a conserved basic amino acid at the P5 position that play an important

processing role, with the cleavage occurring between two small neutral amino acids

(Bonilla et al., 1997).

Based on sequence characteristics of both cleavage sites, Bonilla et al. (1997)

searched the polyprotein sequence for the presence of the consensus cleavage sequence

P5-(R,K)xxx(G, A) I (G, A, V)-PI'. They found another putative cleavage site that would

produce a protein that corresponded with the observed 50 KDa (p50) protein in infected

cells. If that cleavage occurs, it might be a way to regulate the polyprotein processing

since the PCS would interrupt the active site of this protease. We searched for other

putative cleavage sites within the CTVL 1 region, but we did not find any sequence

similar to the PCS 1 or PCS2 positions or to the reported "conserved" closterovirus

cleavage site sequence P2(bulky)P I (G)XXXP4'(negatively charged) reported by Jelkman

et al., (1997). A further option is the possibility of more cleavage sites for CTV that have

not been reported within the polyprotein or other viral/cellular proteins. Studies that










spanned the 400 KDa of MHV ORF 1 a evidenced the presence of other cleavage sites

within the polyprotein of this virus (Schiller et al., 1998). This led to the detection of the

activity of a second putative cysteine protease domain, which had been previously

undetected. PLP2 has a substrate downstream from its physical location in the

polyprotein, and it has different substrate determinants than those of PLP 1, suggesting

that the overall conformation of the protein might affect the efficiency of the processing

that could regulate different activities of the replicase complex (Kanjanahaluethai and

Baker, 2000). Infectious bronchitis virus (IBV) a member of the Coronaviridae family,

also contains two cysteine protease domains. Based on the complete viral sequence

(Boursnell et al., 1987), two papain-like cysteine proteases and a picomavirus 3C-like

protease domain were identified (Gorbalenya et al., 1989). In vitro transcription and

translation of the ORF L a sequence offered the first evidence of proteolytic activity when

an 87 KDa (p87) protein was released from the polyprotein. The molecular mass of this

protein corresponded to that of a protein observed in infected Vero cells (Liu et al., 1995).

This p87 was determined to be the product of the activity of two overlapping papain-like

proteases (Liu et al., 1995). Further characterization of this activity revealed that only the

first proteolytic domain was responsible for the p85 release, and the catalytic amino acids

were mapped to C274 and His437 and the cleavage site to Gly673-Gly674 (Lin and Liu,

1998). When the activity of IBV PLPD- 1 was compared to that of the human coronavirus

229 PLPD and mouse hepatitis virus strains JHM and A59 in terms of the composition of

the catalytic residues and the cleavage specificity, there were similarities between the

proteins, but there also were three major differences. These included no protease activity










for IBV PLPD-1 when expressed in an in vitro system, a different specificity for the

cleavage site recognition at the P5 position where a valine instead of a basic amino acid

was located for the IBV sequence, and the final difference found for the IBV protein was

the lack of trans cleavage for both in vivo and in vitro assays (Lin and Liu, 1998).

Another activity for the IBV protease was the cleavage of a second dipeptide bond

between Gly2265-Gly2266. Both cleavage products were identified in IBV infected cells

(Lim et al., 2000). When the activity of PLP I was studied in human coronavirus 229E

another member of the Coronaviridae family, the activity was similar to that described

for the murine hepatitis virus. MHV cleaves at G141_V241 to form p28, and HCV produces

p9 after a cleavage at Gly"1-Asn"2 reflecting different positions of the cleavage sites. In

contrast with MHV, HCV229E PCP 1 does not mediate more cleavages within the

replicase complex, and it shows trans activity (Herold et al., 1998).














CHAPTER 4
CIS TRANS ACTIVITY OF CTVL 1 AND CTVL2

Cis-proteolysis occurs when the activity is performed in an intramolecular

fashion, whereas trans-activity refers to intermolecular reactions. These and other

characteristics have been used to classify viral cysteine proteases into leader or main

proteases (Gorbalenya et al., 1991; Chen et al., 1996). An initial classification of leader

proteases included those proteases from poty-, bymo- and aphtoviruses. The general

characteristics of these proteases were that they mediated a single cleavage event at their

own C-terminus, being described as accessory proteases. The other group, the main

proteases, encompassed those from a- and rubiviruses. This class of proteases represents

cases in which a single enzyme is capable of performing several or all of the cleavages

during the processing of the polyprotein (Gorbalenya et al., 1991).

Semliki forest virus (Alphavirus, Togaviridae) encodes a main cysteine protease,

nsP2A, which is the only protease needed to process the SFV P1234 polyprotein. The

maturation pathway for this polyprotein is a series of proteolytic processing that includes

both cis only and trans only reactions mediated by nsP2 at specific cleavage sites (Merits

et al., 2001).

Murine hepatitis virus (MHV, Coronaviridae) encodes two papain-like proteases,

PLP-1 and PLP-2. Characterization of the activity of PLP-1 revealed that this protein is

capable of cleaving at different positions of the polyprotein and possesses the ability to










cleave in trans, being compared to the activities presented by the alphavirus nsP2

protease (Bonilla et al., 1997). This characteristics showed strong dependance on the

incubation temperature and the size of the substrates presented to the protease, where low

temperatures and larger substrates resulted in more efficient cleavages, evidencing the

importance of the overall folding of the polyprotein in the process (Teng et al., 1999).

The other protease, PLP-2, encoded in the MHV genome also has been demonstrated to

be a trans-active protease, having its substrate site more than 1000 residues downstream

from its active site (Kanjanahaluethai and Baker, 2000). These two proteases showed

characteristics of main cysteine proteases.

Besides the proteolytic domain, an "X" domain was found, exclusively for the

main proteases (Gorbalenya et al., 1991). This domain was later related with the trans-

proteolytic activity of RUB NS protease (Liang et al., 2000). Other examples of main

proteases include proteases encoded by foot and mouth disease virus (Kirchweger et al.,

1994) and rubella virus (Liu et al., 1998). In all the cases, these enzymes cleave both in

cis and trans and in different sites apart from the catalytic center within the polyprotein.

There are several examples of leader proteases encoded in the genomes of

different viral groups. In the translation of the major large dsRNA of the hypovirulence

associated virus of the chestnut blight fungus it was found that a 29 KDa protein was

released by cotranslational proteolysis. This proteolytic activity was associated with the

p29 protein, and its sequence characteristics had similarities with the potyviral HC pro

(Choi et al., 199 1b) relating it to the leader proteases (Choi et al., 1991 a).










For the arterivirus replicase ORFIa protein there have been described either one

PLP for equine arteritis virus (EAV) or two PLP domains, PCPa and PCP3 for lactate

dehydrogenase elevating virus (LDV) and porcine reproductive and respiratory virus

(PRRSV). In either case, the proteases released themselves from the polyprotein in a cis

autocatalytic reaction and did not show any further processing of the polyprotein (Den

Boon et al., 1995).

Even though there is some variability among the characteristics of the viral

proteases, there are features that are constant among them. Within the order Nidovirales,

the family Artriviridae and Coronaviridae contain both main and accessory proteases.

The accessory proteases share characteristics as the recognition of 1 or 2 cleavage sites,

and they are located at the N-terminal half of the polyprotein. There is at least one small

amino acid at the scissile bond, and there are cysteine and a histidine residues at the

catalytic dyad (Ziebuhr et al., 2000).

Besides the common characteristics of the accessory proteases for this order, there

are specific attributes for each family. The arterivirus accessory proteases have a short

spacer distance between their catalytic residues, which is similar to those found in other

PCPs (Gorbalenya et al., 1991); they are located at an amino terminal position within the

polyprotein; and they cleave downstream of their catalytic domain (Ziebuhr et al., 2000).

Examples of these proteases are found in EAV, LDV and PRRSV (Den Boon et al.,

1991). The coronavirus accessory proteases have a spacer between their catalytic

residues that is almost as twice as long as those of the arteriviruses. The proteases are

separated by at least 1000 residues, which gives them a more central location within the










polyprotein, and they perform proteolytic cleavages upstream of the catalytic domain.

Viruses that encode proteases in this group include murine hepatitis virus (MHV), human

coronavirus (HcoV), transmissible gastroenteritis virus (TGEV) and infectious bronchitis

virus (IBV) (Ziebuhr et al., 2000).

Materials and Methods

Virus Isolate, cDNA Synthesis and Cloning

The CTV strain and general techniques were described in Chapter 1.

Descriptions of the cDNA constructs used to demonstrate cis and trans activity of CTVL 1

and CTVL2 are found in Chapters 2 and 3.

Post Translational Cis/Trans Activity Assays

In vitro transcription and translation was performed as described in Chapter 2.

Constructs used as substrate were translated in the presence of [3H]Leu. Constructs used

to synthesize enzymes were translated in the presence of a complete amino acid mix with

no radio-labeled amino acid present. After the reaction was completed, RNase was added

to the reactions, and equal amounts of enzyme and substrate were mixed and incubated

for an additional hour under the same conditions. The reactions were performed at both

22 and 30 'C. Following the incubation, the samples were analyzed by SDS-PAGE as

previously described.

Co-Translational Cis/Trans Activity

Enzyme and substrate cDNA clones were co-translated in the same reaction mix.

The reaction conditions were those used for the single translations, except that both

plasmid DNAs that were being tested were added.











Results

CTVL 1 Encodes a Cis Acting Protease Tested in an In vitro Assay

The experimental design that we used to demonstrate trans activity of CTVL 1 and

CTVL2 included the production of inactivated proteases by SDM of an amino acid in the

active site as described in chapter 3. The inactive proteins were then used as substrates to

demonstrate complementation in trans of the proteolytic reactions when incubated in the

presence of an active protease. The in vitro transcription and translation reactions to

produce the substrate proteins were performed in the presence of a radio-labeled amino

acid, whereas those to be tested for enzyme activity were synthesized in the absence of

the radio-labeled amino acid.

We previously demonstrated that the translation product of CTVL 1 -PCS 1 yielded

both full length size protein of 59 KDa and a mature protein of 54 KDa, evidencing the

autoproteolytic cleavage at the PCS I, mediated by CTVL 1. Figure 4.4.1B shows a

complementation test in which we used unlabeled CTVL 1-PCS I as the source of the

proteolytic activity. To evaluate CTVL 1 trans activity at the PCS 1, we tested the

substrate site in the PCS 1 -CTVL2-PCS2 construct. Previously we showed that this

construct was proteolytically inactive at the PCS 1. Therefore, if it showed any

processing, the proteolytic activity must originate from the trans supplemented enzyme.

CTVL 1 failed to cleave the PCS I when tested in trans under these conditions.

Since processing of PCS 1 in the PCS I -CTVL2-PCS2 construct was never

observed, there was no evidence that this site was correctly folded when presented in this

construct. This may be the reason for the trans cleavage failure. To evaluate this










possibility we used the CTVL lm-PCS I construct, which basically is the inactivated

version of the protease that was in the assay. The result of this experiment also did not

indicate trans proteolytic activity of CTVL 1 (Figure 4.1 B, lanes 5 and 6).

Additional tests of trans proteolytic activity for CTVL 1 used the CTVL I m-L2-

PCS2 translation product as a substrate. This protein represents approximately the first

110 KDa of the CTV ORFI N-terminal region, including both proteolytic domains and

putative cleavage sites for these proteases. Trans complementation with the CTVL 1-PCS

translation product did not change the molecular mass of the protein as shown in Figure

4. 1D, lane 1. To confirm that the unlabeled enzyme was being translated, an aliquot of

the unlabeled translation reaction was incubated in the presence of [3H]Leu. The result is

shown in Figure 4.1 D, lane 3, corroborating the synthesis of the active protease.

CTVL 1 -L2-PCS2 did not Complement the Proteolytic Reaction in a Post-Translational
Assay.
The translation product of CTVL I -L2-PCS2 was shown in Chapter 3 to have

proteolytic activity and caused at least 3 cleavages within the region. To test if any of the

remaining mature proteins had trans proteolytic activity we tested these enzymes with all

the available constructs that carried any of the substrate sites.

Figure 4.2 shows the results of incubations with the PCS I -CTVL2-PCS2 as

enzyme and, CTVL I-PCS 1, CTVL I m-PCS I and CTVL I m-L2-PCS2 as substrates. In

none of the cases tested did incubation with the unlabeled proteolytic active product

change the electrophoretic pattern observed for both substrates.













60


A B


G46 G4076 0937


2 3 4 5 6
Crvu I. jj 59-54- kfl
C403S/Ai


~1CrVLI PCSI S SkD. 7


--13

NUMBER ENZ*AME SUBSTRATE
L PCS1-CTIL2-PCS2
2 CTVLI-PCSI PCSI-CTVLI-PCS2
-46
3 CTVL.PS1
4 CTVLI.PCS1 CTVLI-PCSI 32
L5 CTVL.I.PCSI
6 CTVLI PCSI CTVLI-PCSI
C D



1 2 3 4 kDa
G4 6 5 006 9G

.216

CTVLI PCS1 39 kD.
-132

03s; 78
CTVLI L2"PCS2 113 kDa


NUMBER CNZYM B rSA1Z
I CTVLI PCS1
2 CTVLI.-L2-PCS2 -46
3 CTVL I -PCS I
4 CIVLI-PCSI CTVL -L2- PCS2 32


Figure 4.1: CTVL I-PCS 1 trans complementation of proteolytic activity. (A) Schematic

representation of the proteins used in the trans-complementation assay shown in panel B.

The putative cleavage sites are indicated. Mutated CTVL 1 -PCS 1 carried either C403A or

C403S substitutions to inactivate its auto-proteolytic activity and was used as a substrate

for the trans reaction. Radio-labeled substrates were incubated for one hour with

unlabeled active enzyme and then subjected to electrophoresis. (B) SDS-PAGE

autoradiogram of in vitro transcription and translation [3H]Leu labeled products as

described for panel A. (C) Schematic representation of CTVL 1-PCS I trans proteolytic

activity assay. Mutated CTVLl-CTVL2-PCS2 carrying either C403A or C403S

substitutions was used as a substrate for trans proteolytic complementation of CTVL 1.

Putative cleavage sites and expected molecular mass of unprocessed products are

indicated. (D) SDS-PAGE Autoradiogram of in vitro transcription and translation

[3H]Leu labeled products as described for panel C. Lane 1 and 2 show the enzyme and

substrate used in the assay, respectively. Lane 3 after mixing the enzyme and the

substrate, unlabeled enzyme was labeled with [3H]Leu to corroborate its translation.

Lane 4 shows the complementation assay as described for panel C.








61

A B
04 1 2 a 4 5 6 7 8


kDa


CTVLI-PCSI 5"- ~ 78
CTVLI.mPCS1 S9 kD.4
SUBSTRATES
PCSI '1- -PCS2 CSll2 A 63 kD.
-YTL -:,-CTVL2 PCS2 I 11 kD.
46





Figure 4.2: CTVL 1 -L2-PCS2 trans proteolytic activity. (A) Schematic representation of
the translation products of the constructs used in the in vitro transcription and translation
assay. (B)SDS-PAGE autoradiogram of the trans proteolytic complementation assay.
Lane 1 and 2 PCS 1-CTVL2-PCS2 with and without the CTVL1-L2LPCS translation
product. Lane 3 5, CTVLI-PCS1, CTVLlm-PCS1 and CTVLlm-L2-PCS2. Lane 6 8
same proteins as in 3 5 supplemented with unlabeled CTVL 1 -L2-PCS2.


This suggests that trans proteolytic activity is not associated with the mature

translation products from the CTVL 1 -L2-PCS2 cDNA construct. So far, we have

evaluated the ability of both CTVL 1 and CTVL2 to perform trans proteolytic reactions

over the known cleavage sites. The enzymes were prepared from different constructs in

all the combinations where we had observed proteolytic activity. Substrates were

presented under conditions that simulated the native conformation or where they were

previously cleaved when the proteolytic domain was active. Although no other changes

were introduced in the substrate proteins apart from the inactivation of the catalytic

domain, we cannot rule out a conformational change that could affect the folding and

hence the recognition of the cleavage site in the trans reaction.


6

2


,.; OM!










CTVL2 may have Trans Activity as Shown by a Co-Translational Trans Proteolytic
Assay

To test trans proteolytic activity we used translation products that we knew were

proteolytically active because they had experienced auto-proteolytic maturation. Thus,

we had done experiments where both protease and substrates were independently

translated and then mixed to assess trans proteolysis in a post-translational fashion. This

approach was a convenient way to study the system because it provided the possibility of

having only the substrate labeled, making the analysis of the results more forthright. The

problem with this approach is that even though it was a clean way to observe the results,

it did not provide any evidence that the proteolytic domains remained active after being

processed, and that might have introduced an artifact into our results.

To further test the absence of trans proteolytic activity of CTVL 1 and CTVL2 we

performed a trans co-translational assay where both enzyme and substrate constructs

were translated together in the same tube, and the patterns observed were compared to

those of the individual constructs while trying to differentiate between superimposition of

patterns or patterns produced by the interaction between the proteins translated from both

constructs. The results of these experiments can be observed in Figure 4.3. Panel A

shows the electrophoretic pattern that each individual cDNA translation product produced

and which has been reported in previous chapters. Lanes I and 2 evaluated the trans-

activity of CTVL 1 when presented from the CTVL 1 -PCS I construct on the CTVL I m-L2-

PCS2 and CTVL 1-L2m-PCS2 substrates. In neither case did we see a different pattern

from that of the superimposition of both translation products, indicating no trans-

proteolytic reaction in the system.








63

Lane 3 and 4 evaluated the trans-activity for both proteases when presented in the

CTVL 1-L2-PCS2 construct. To discriminate between any possible trans-activity of

either protease contained in this construct, we used as a substrate the CTVL1-L2-PCS2

constructs with mutations in either CTVL 1 or CTVL2. Trans-activity in this case would

be evidenced by the complementation of the particular mutation, producing the expected

wild type electrophoretic pattern. Under these conditions, Figure 4.3B lane 3 shows the

complementation experiment to evaluate the trans activity of CTVL 1. This experiment,

as with the previous ones done using CTVL 1 as the enzyme, did not show any evidence

of trans activity, indicating that this protease is probably a cis acting protein only.

The use of the CTVL 1-L2m-PCS2 construct allowed us to evaluate the trans

activity of CTVL2 when supplemented as part of the CTVL1-L2-PCS2 proteolytic

construct. Under these conditions we were able to obtain some evidence suggesting that

CTVL2 might be a trans-acting protein at the unpredicted cleavage site. Even though the

experimental design did not allow us to discriminate between the bands that corresponded

to each of the cDNA translation products, the major processing product corresponds to

that of the pattern produced when both proteases are active in the construct (Figure 4.3B

lane 4). Based on this result, it seems that the pattern observed is different from the result

of superimposition of the individual translation products for each construct in the

reaction. When this result is compared with that of the other experiment where we used

the same substrate but complementation did not occur (Figure 4.3B lane 2), we can see

that the typical pattern produced by the inactivation of CTVL2 is reduced for the trans-

complemented reaction.


















E S
Lane 1


E S
Lane 2


Enzyme: CTVL1-PCS 1
Substrate: CTVLlm-CTVL2-PCS2


ES


Lane 3


Enzyme: CTVL1-CTVL2-PCS2
Substrate: CTVLlm-CTVL2-PCS2


Enzyme: CTVL1-PCS1
Substrate: CTVL1-CTVL2m-PCS2


E S


Lane 4


Enzyme: CTVL1-CTVL2-PCS1
Substrate: CTVL1-CTVL2m-PCS2


1 2 3 4 kDa


-132

- 78


-59
--- 54
m m --- 52


- 46

- 32


PI-SI
C403$/A
PI-SI


C403S/A

PIm PU-SH
C403S/A
PI-P]IM SII


Figure 4.3: Assessment of cis-trans activity of CTVLI and CTVL2. (A) SDS-PAGE
autoradiograms of [3H]Leu labeled wheat germ coupled in vitro transcription and
translation reactions of cDNA constructs used to test cis-trans activities of CTVL 1 and
CTVL2. (B) Co-translation of enzyme substrate cDNA constructs described in part A.
(C) Schematic representation of the cDNA constructs used in these experiment.


QM -


- -
own


59-.44 kDa


,9 kDa

113 kDa

113 kDa

113 kDa


I


G44 G,465 G976 Gg77
4 + l 4llllllllll










Discussion

The purpose of this study was to determine the possible cis-trans characteristics of

both CTVL1 and CTVL2. The nature of the system allowed a simpler experimental

design to test for CTVL I activity, since it seemed to be independent of other proteolytic

reactions within the polyprotein. For CTVL2 we have already determined that it requires

CTVL 1 activity to trigger a second cleavage within the CTVL 1 region. Therefore, in

order to be able to evaluate this activity, both proteins had to be expressed together.

To assess these characteristics for either protease we tried to study them as

individual proteins (CTVL 1) or as a region with different proteolytic activities (CTVLI-

L2-PCS2). Trans-activity of CTVL I was evaluated in different ways by attempting to

simulate the conditions where the cleavage occurs at PCS 1. As enzyme sources we used

constructs for which we had previously observed proteolytic activity (CTVL1-PCS1 and

CTVL1-L2-PCS2). The substrates used in the assay included PCS 1-CTVL2-PCS2,

CTVL I m-PCS I and CTVL 1 m-CTVL2-PCS2. Complementation of CTVL 1 proteolytic

activity on the PCS 1 was not observed for any of the combinations tested. This may

indicate that CTVL 1 is a cis-acting protease which releases itself from the polyprotein, a

characteristic of all the described leader proteases.

Even though we have not been able to demonstrate trans-activity for CTVL 1,

there are many factors that could have influenced the result. In the characterization of the

trans-activity of a papain-like protease of the marine coronavirus MHV, it was found that

the PLP- 1 protein that had been previously described as cis acting only (Baker et al.,

1989) was able to act in trans as well (Bonilla et al., 1997). The newly described trans-










activity for this protease was evidenced by the expression of deletion constructs of the

substrate. This suggested that the observed trans activity was due to the increased

availability of the substrate to the trans activity of PLP- 1 protease, rather than the product

of altered catalytic properties of the enzyme (Bonilla et al., 1997).

More evidence of the importance of protein folding in the trans activity of PLP-1

was obtained though a series of experiments performed at different temperatures. Trans-

proteolysis at two cleavage sites was significatively more efficient when performed at

22C rather than 30 'C. Further observation revealed a more efficient cleavage at 16 'C,

and reactions performed at 37 'C were even less efficient than those performed at 30 'C.

It has been suggested that this temperature dependence for trans-cleavage could be a

requirement for a specific folding of the polyprotein for the recognition of the trans-

cleavage sites, and that it is achieved only at low temperatures in an in vitro assay (Teng

et al., 1999). To test temperature dependence in our system, we performed the trans

complementation reactions at both 22 'C and 30 'C. However, we did not observe any

differences under these temperature conditions, with the CTVL 1 reaction being cis-acting

only under both temperature conditions tested.

PLPD- I from the coronavirus avian infectious bronchitis virus (IBV) has been

demonstrated to be a cis-acting only protease when it is expressed in vitro in rabbit

reticulocyte lysates. When the protein is expressed in intact cells, it can act in trans on

the same substrate (Lim et al., 2000). The trans-activity of CTVL 1 cannot be properly

evaluated in vivo due to the unavailability of an infectious cDNA clone in our laboratory,

which would allow us to perform in vivo complementation assays for these reactions.








67
Another factor which needs to be taken into consideration when analyzing the cis-

trans proteolytic activity of these proteases is that they are being expressed in the N-

terminus of the polyprotein in an in vitro translation system. The length of the substrate

was demonstrated to be important for the trans-activity of MHV-A59 PLP-1. When

different sizes of the substrate were tested, it was found that no proteolytic activity was

detected with substrates smaller than 301 residues, and that approximately 44% of the

protein was processed when the 622 residue size was achieved, reaching maximum

processing at the 867 amino acid length (Teng et al., 1999).

Another example which demonstrates the importance of the substrate

conformation in a trans-proteolytic reaction, is the rubella virus (RV) protease. Co-

expression of a construct of approximately 2000 amino acids which included the

proteolytic region bearing mutations at the cleavage site to serve as a protease and a

construct with mutations at the active site to serve as a substrate did not reveal trans-

cleavage for this protein (Chen et al., 1996). However, when the whole ORFI of RV was

expressed, it was demonstrated that the cysteine protease encoded within it was capable

of performing proteolytic cleavages both in cis and in trans, suggesting that either

conformational changes of smaller constructs or the requirement for an intact ORF I

translation product could explain previous failures to detect trans proteolysis (Yao et al.,

1998).

Our experimental system only represents an in vitro translation of 1016 N-

terminal amino acids of the possible 3124 residues encoded in CTV ORF l a. To date we

do not know about other possible cleavage sites within the polyprotein or the effects of









68
expressing these proteins out of the polyprotein context. There is some evidence that the

polyprotein might undergo further proteolytic processing in vivo, due to the presence of

free helicase and methyl transferase domains in BYV infected tissues (Erokhina et al.,

2000) and free RdRp in CTV infected tissue (1evik, personal communication). The

origin of the protease activities responsible for these cleavages remains to be determined.

With our current information on CTVL 1 and CTVL2 we can not reach any conclusion

about the involvement of these proteins in those proteolytic reactions. Other proteases

have been described whose characteristic do not agree completely with the description of

the leader proteases. For blueberry scorch carlavirus and turnip yellow mosaic virus, the

cysteine proteases involved in the processing of the polyproteins are in both cases cis-

acting only enzymes as the leader proteases, but their substrates are located

approximately 400 amino acids downstream from the catalytic dyad (Bransom and

Dreher, 1994; Lawrence et al., 1995), contrasting with the typical 40 residues found for L

proteases (Gorbalenya et al., 199 1). CTV proteases have been predicted to be a

duplicated papain-like leader protease similar to L-pro from BYV (Karasev, et al., 1995).

The results of the co-translational trans proteolytic assay did not reveal any evidence of

trans activity for CTVL 1. The results observed in the complementation of CTVL2

activity suggest that this protease its able to cleave its substrate in trans. If CTVL2 its

confirmed to be a trans active protein, then its classification as a leader protease should

be reconsidered.













CHAPTER 5
SUMMARY AND CONCLUSIONS


The N-terminal region of citrus tristeza closterovirus ORF1 strain T2K was cloned

and used for in vitro transcription and translation assays to determine the mechanism of

proteolytic processing in the region. From the results obtained, there are at least three

proteolytic cleavages in the region, under the conditions studied. These proteolytic

activities were insensitive to a broad spectrum protease inhibitor cocktail as well as to the

specific cysteine protease inhibitors E64 and N-ethylmaleimide.

The putative catalytic amino acids C"3- H464 and C896- H956 were experimentally

confirmed by site directed mutagenesis as the active residues of CTVL1 and CTVL2,

respectively. The importance of the amino acid that occupies the P1 position in both

putative cleavage sites was established as well, being determined that changes in this

position were not well tolerated by the system, resulting in inhibition or reduction of the

proteolytic processing of the area.

CTVL1 cotranslationally cleaved itself from the rest of the polyprotein at the

PCS 1. Cis-trans complementation assays failed to demonstrate trans activity for this

protein under the conditions tested. These results jointly with the sequence characteristics

such as the relative position of the active amino acids between each other and within the

proteolytic domain, as well as the position of the cleavage site for this protease, suggested

the CTVL1 might belong to the leader proteases group. CTVL2 was able to

69











cotranslationally release itself from the polyprotein cleaving at the PCS2. This activity

was dependent on the length of the protein since it was not observed until the CTVL1-L2-

PCS2 was expressed as a unit. Also it was demonstrated that this cleavage was

independent of the CTVL1 activity since it also occurred when the first proteolytic

domain was inactivated.

A second activity was described for CTVL2, hydrolysis of a cleavage site within

the area of CTVL1. This activity was CTVLl-dependant since inactivation of this protein

eliminated the second cleavage performed by CTVL2. From cis-trans complementation

assays, co-translation of the substrate and the enzyme constructs suggested that this

second activity of CTVL2 occurs in trans. This latest characteristic does not agree with

the definition of a typical leader protease.

With these data we developed a working model in which CTVL1 and CTVL2 co-

translationally release themselves from the polyprotein in a cis acting proteolytic function.

The release of both proteases allow CTVL2 to act in trans in a region within the area of

CTVL1. Sequencing of the proteolytic products is necessary to confirm the exact location

of this cleavage site, and its biological significance remains to be determined. Processed

proteins may perform other proteolytic or non-proteolytic activities in the cell during the

course of the viral infection.

The biological significance of these studies performed in vitro remains to be

confirmed in an in vivo system using an infectious clone of CTV.













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BIOGRAPHICAL SKETCH

Jorge Vdzquez-Ortiz was born in Caracas, Venezuela, in 1970. He obtained a

bachelor's degree in biology in 1994 from the "Universidad Central de Venezuela" in

Caracas. After his graduation he worked at the "Universidad Central de Venezuela" as an

assistant professor in the human physiology department, medical school. In 1996 he

started his studies for a Ph.D. in plant molecular and cellular biology at the University of

Florida, from which he graduated in 2001.








I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




C. Niblett, Chair
Professor of Plant Molecular and Cellular
Biology


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




B. M. Dunn
Distinguished Professor of Biochemistry
and Molecular Biology


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




J. M. avis
Associate Professor of Forest Resources and
Conservation


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acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




R.F. Lee
Professor of Plant Pathology









I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




G. Moore
Professor of Horticultural Science


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




M. L. Wayne
Assistant Professor of Zoology






This dissertation was submitted to the Graduate Faculty of the College of
Agricultural and Life Sciences and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.



August, 2001



697
Dean, College of Agricult1 d Life
Sciences


Dean, Graduate School



































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