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Different replication requirements in the homologous 3' ends of a positive strand rna virus and its subviral RNA

Dissertation
Author: Rong Guo
Abstract:
SatC is a noncoding subviral RNA associated with Turnip Crinkle Virus (TCV), a small (4054 nt) single-stranded (+)-strand RNA virus belonging to the Carmovirus genus. Because of its small size (356 nt) and TCV-derived 3' end, satC has been successfully used as a model to elucidate sequence and structural requirements for TCV RNA replication. Although satC is considered a model to identify cis-acting elements required for TCV replication, recent findings indicate distinct differences in structures and functions of these related sequences. RNA2D3D predicts that part of the TCV 3' end (H5, H4a, H4b and two pseudoknots) folds into an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits and is required for translation. SatC contains a similar 3' end with 6 nt differences in the 100 nt TSS region. RNA2D3D did not predict a similar structure for satC TSS region, and satC did not bind yeast ribosomes. satC nucleotides were changed into TCV TSS bases to determine which base differences are responsible for the loss of the TSS in satC. Changing these bases all increased ribosome binding but surprisingly none of them had an effect on satC accumulation in protoplasts and plants. Therefore satC may need these and other 3' end base differences for its required conformational switch for efficient replication, and not to inhibit ribosome binding. In vivo genetic selection (SELEX) of the linker sequence between H5 and the Pr showed the conservation of UCC, which led to the discovery of Ψ2 . Ψ2 is required for both viral and satC accumulation in protoplasts. H5-Pr linker had no significant structural change after RdRp binding in satC, which is different with TCV H5-Pr linker. TCV H5-Pr linker had a major structural change upon RdRp binding, and is proposed to be involved in a conformational switch. Replacement of satC H4a with randomized sequence and scoring for fitness in plants by SELEX resulted in winning sequences that contain an H4a-like stem-loop. SELEX of H4a/H4b in satC generated two different structures: wt H4a/H4b-like structure and a single hairpin structure. Two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. With the protection of CP, satC can form higher amount of dimers that have additional nucleotides at the junction sites in the absence of TCV. The extra nucleotides are not necessarily associated with an active TCV RdRp.

TABLE OF CONTENTS

Section

Page

ACKNOWLEDGEMENTS ……………………………………………………...

i i

TABLE OF CONTENTS…………………………………………………… .. …

i v

LIST OF FIGURES………………………………………………………… ... ....

ix

LIST OF TABLES………………………………………………………….… ... .

xi

ABBREVIATIONS………………………………………………………… …. ..

x ii

CHAPTER I: Elements Involved i n Replication of Positive Strand RNA

Viruses …………………………………………………………………………...

1

Introduction………………………………………………………… ….. …

1

Subviral RNAs A ssociated W ith P ositive S trand RNA V iruses …… …... …

7

Origins of subviral RNAs ………………… ……………………… ..

7

Relationship between subviral RNAs and their helper viruses …. .. ..

8

Subviral RNAs as model systems ……………………………. .. …..

1 1

Basic Aspects of Virus Replication.………………………………………..

1 3

Model Systems for Studying Virus Replication…………………………....

1 6

TBSV

replication……………………………………………… ….. .

1 6

BMV

replication ……………………………………………… … …

1 8

TCV replication………………………………………………… .. ...

20

Structural comparison between satC and TCV ……… …… ...

2 5

Conformational changes …………………………..……… . ..

30

Thesis plan……………………………………………………………… . ...

32

CHAPTER II: The D ifference B etween S atC and TCV I n T he TSS R egion ……

3 3

v

Introduction...................................................................................................

3 3

Materials and Methods ……………………………………………………..

34

Construction of satC mutants ……………………………… …... .…

3 4

In vitro

RNA synthesis using T7 polymerase ………………..… .. ...

3 5

Small - scale plasmid DNA isolation (Alkaline Lysis) ………… .. .....

3 6

Small - scale plasmid DNA isolation (STET) ……………… …… ….

3 6

Protoplast preparation and inoculation …………………………......

3 7

Extraction of total RNA from Arabidopsis

protoplasts ………….…

38

Northern blotting using RNA gels ………………………………....

39

Plant growth and inoculations ………………………………….......

40

Competition in Plants ………………………………………………

40

Small - scale viral RNA extraction from infected turnip plants ……..

41

Cloning of viral progeny into pUC19 ………………………..….....

41

RNA in - line probing …………………………………………….....

42

Results ……………………………………………………….………….....

4 6

Ψ 3

is not required for satC accumulation in protoplasts …………....

4 6

DR has some flexibility in its location ……………………………..

4 8

Ribosome binding was enhanced in satC mutants ………………....

5 0

Enhanced ribosome binding to satC does not impact satRNA replication …………………………………………………………..

55

Enhanced ribosome binding to satC does not impact on satRNA movement in planta …………………………………….…….…….

55

Alterations of satC into TCV TSS affect the structure of Ψ 2

and

vi

H4a l oop ……………………………………………………………

58

Discussion...………………………………………………………………..

61

CHAPTER III: The importance of a flexible linker region in satC replication ….

66

Introduction...................................................................................................

66

Materials and Methods……………………………………………………..

73

In vivo

SELEX …………………………………………………..…

7 3

Construction of satC mutants ……………………………………....

7 3

In vitro

transcription, inoculation of Arabidopsis

protoplasts, and Northern blots …………………………………………………..…..

74

Purification of p88 from E. coli …………………………………....

7 5

RNA DMS modification and primer extension …………………….

7 6

Filter binding assay ……………………………………………..….

7 7

Results ……………………………………………………….………….....

80

The H5 - Pr - linker has sequence flexibility ………………………....

8 0

UCC is conserved in the linker region, and forms Ψ 2 ………….….

8 4

The conserved AACC does not base pair with the 5' end …………

8 4

The linker region is not directly interacting with H5 lower stem ……………………………………………………………...…

88

Structure of satC H5 - Pr linker remains unchanged upon RdRp binding.………………….………………………………………....

88

In vitro

filter binding assay showed the importance of the Pr and the DR for RdRp binding……………………………………….….

91

Discussion……………………………………………………………….…

9 2

vii

CHAPTER IV: The R elationship B etween H4a A nd H4b I n S atC …………...…

9 6

Introduction...................................................................................................

9 6

Materials and Methods……………………………………………………..

98

Construction of satC mutants ………………………………........…

9 8

In vivo

SELEX (H4a/H4b)……………... .........................................

9 8

Four way in vivo

SELEX……………………… ………………... ...

1 0 0

Accumulation of viral RNAs in protoplasts …………………..……

1 0 1

Results ……………………………………………………….………….....

104

SatC H4a SELEX results in retention of a stem - loop ……………...

1 0 4

Testing possible interaction between H4a and H4b ………………..

1 09

Two distinctive functional structures result from in vivo

SELEX of satC H4a/H4b ……………………………………………..………..

111

Self - evolution of three H4a/H4b SELEX 3 rd

winners ……………...

1 1 5

Ψ 2 is confirmed in H4a/H4b SELEX derived two distinctive functional structures ………………………………………………..

118

Ψ 2

is n ot d irectly i nteracting w ith

the DR ……………………….…

1 18

The DR may not interact with an upstream satD - derived sequenc e.

1 2 4

Discussion...………………………………………………………………..

1 2 6

CHAPTER V: The formation of satC dimers in the absence of TCV RdRp …….

1 3 0

Introduction...................................................................................................

1 3 0

Materials and Methods……………………………………………………..

131

Construction of T - DNA - based plasmids for agroinfiltration ……....

1 3 1

Agroinfiltration procedure …………………………………….........

1 3 3

viii

RNA extraction and Northern blots …………………………….….

1 3 3

RT - PCR and cloning ……………………………………………….

1 3 4

Protein extraction from plants and Western blotting analysis ……...

1 3 5

Results ……………………………………………………….………….....

140

Successful expression of TCV system by agroinfiltration …………

1 40

The requirement of CP for satC expression in

the absence of active TCV RdRp …………………………………………………………

140

The effects of other silencing suppressors for satC expression in the absence of TCV ………………………………………………...

142

SatC can form dimers in the absence of active TCV RdRp ……..…

1 4 4

Dimer junction sequence …………………………………………...

1 4 6

Discussion...…………………………………………………………..........

1 4 6

APPENDIX …………………………………………………… …………………

1 49

CONCLUSIONS… ………………………………………………………………

15 2

REFERENCES …………………………………………………………………..

1 57

ix

LIST OF FIGURES

Figure

Page

Figure 1.1 TCV genomic RNA and associated subviral RNAs ……………… ….

2 2

Figure 1. 2

MPGAfold - predicted TCV and satC 3' terminal structures ………… ..

2 3

Figure 2 . 1

Analysis of a predicted pseudoknot between H4a and

the DR region ..

4 7

Figure 2.2 Analysis of the position flexibility of DR ………………………… . …

49

Figure 2. 3

Analysis of satC mutants which contain TCV TSS sequence

…… .. ...

5 1

Figure 2. 4

Replication of satC mutants which contain TCV TSS sequence

… ….

5 3

Figure 2. 5

In - line probing of 5 '

half of wt satC and var ious mutants … …………

59

Figure 2. 6

In - line probing of 3 '

half of wt satC and various mutants ………… …

6 0

Figure 3.1 m F old predicted satC structures showing three different

conformations for H5- Pr - linker.……………………..………………..

68 Figure 3.2 In vivo selection of H5 - Pr linker region. …………………………… .. .

81

Figure 3.3 Analysis of H5 - Pr linker region

SELEX ………… .. …………… . …. ..

8 2

Figure 3.4 UCC is involved in Ψ 2 ……………………………………….. … ……

8 5

Figure 3. 5

Conserved AACC is not interacting with the 5' end……………. … .. ..

8 7

Figure 3. 6

DMS structure probing of in vitro transcribed satC with or without

TCV RdRp ……………………………………………………………

90 Figure 4.1 H4a and H4a/H4b in vivo SELEX ……………………………… …. ...

1 0 5

Figure 4.2 H4a

in vivo

SELEX results in retention of a stem - loop ……… . ……..

1 0 8

Figure 4.3 Two potential conformations of satC H4a and H4b. ………………..

1 1 0

Figure 4.4 Possible structures adopted by the H4a/H4b SELEX winners……….

11 4

Figure 4. 5

Structure probing of satC transcript s with and without compensatory

x

mutations in H5. .………………………………………… … …….. …

1 2 2

Figure 4.5 P ssible interaction s

between DR and region X. ..……………… …… ..

1 25

Figure 5.1

Physical characteristics of T - DNA constructs of TCV and satC

RNAs ………………………………………………………………….

138 Figure 5.2 The procedure of agroinfiltration.

…………….… ……… ……… …...

1 4 1

Figure 5.3 SatRNA expression in the presence of CP. …………………… . …….

1 4 3

Figure 5.4 Junction sequences of the dimers.

…………………………… …... .…

1 4 5

Figure A.1 Putative interaction between satC and TCV …………………………

15 1

xi

LIST OF TABLES

Table

Page

Table 1.1 Cellular membrane associated with plant positive strand RNA viruses

6

Table 2.1 S atC mutants used in Chapter II…………………… ………… .. …. ….

4 4

Table 2.2 O ligonucleotides used in Chapter II …………………………………...

4 5

Table 2.3 Ribosome binding and accumulation in protoplasts of satC mutants …

54

Table 2 . 4

SatC sequences obtained from the competition among the satC

m utants in plants ……………………………………………………….

57 Table 3.1 Carmoviruses H5 - Pr - linker sequences………………… …………… ...

7 2

Table 3.2 Constructs

used in Chapter III……………………… ……… . …… …..

78

Table 3.3 O ligonucleotides used in Chapter III……… …………… ….……. …...

79

Table 3.4

Summary of in vivo SELEX of H5 and Pr linker ……… …………… ..

8 3

T able 3.5 RdRp binding of satC fragments …………………………… ……… …

9 2

Table 4.1 Constructs

used in Chapter IV……………………………… ... …… …

1 0 2

Table 4.2 O ligonucleotides used in Chapter IV………………… ……………….

1 0 3

Table 4.3 SatC sequences obtained from H4a SELEX rounds 1, 2, 3, and 5… ... .

1 0 7

Table 4.4 SatC sequences obtained from H4a/H4b SELEX rounds 1,2,3,and 5 ...

1 1 2

Table 4.5 H4a/H4b SELEX winner Q ab

based derivatives……………… ….. .. …

1 1 6

Table 4.6 SatC sequences obtained from self - evolution of satC H4a/H4b

SELEX 3 rd round winners Kab and Xabs…………….……………….

117 Table 4 .7

SatC Four Way SELEX …………….…… ………………………… …

1 2 3

Table 5.1 Constructs

used in Chapter V………………………… …… . ……….. .

1 39

Table 5.2 O ligonucleotides used in Chapter V………….… ……… …. … . ……...

14 0

xii

LIST OF ABBREVIATIONS

%: Percent

°C: Degrees in Celsius

2,4 D: 2,4- Dichlorophenoxyacetic acid

3’PE: 3’ - Proximal element

5’PE: 5’ - Proximal element

ATP: Adenosine triphosphate

b: Base(s)

BMV:

Brome mosaic virus

BaMV: Bamboo mosaic virus

BBSV: Beet black scorch virus

bp: Basepair(s)

BSA: Bovine serum albumin

BVDV: Bovine viral diarrhea virus

BYDV: Barley yellow dwarf virus

CarMV: Carnation mottle virus

CCFV: Cardamine chlorotic fleck virus

CCS: Carmovirus consensus sequence

CIRV: C arnation Italian ringspot virus

CMV: Cucumber mosaic virus

CNV: Cucumber necrosis virus

CP: Coat protein

CPMoV: Cowpea mottle virus

xiii

CTP: Cytidine triphosphate

CyRSV: Cymbidium ringspot virus

D: Dimer

DI RNA: Def ective interfering RNA

diG: Defective interfering RNA G

DNA: Deoxyribonucleic acid

DNA: Deoxyribonucleic acid

dNTP:

Deoxynucleotide triphosphate

dpi: Days post inoculation DR: Derepressor element in satC

DTT: Dithiothreitol

DV: Dengue virus

E. coli: Escherichia coli

EAV: Equine arteritis virus

EDTA: Ethylene diamine tetraacetic acid , disodium, dihydrate

eEF1A: Eukaryotic elongation factor 1A

eIF(iso)4E: Plant isoform of eIF4E

eIF(iso)4F: Plant isoform of eIF4F

eIF(iso)4G: Plant isoform of eIF4G

eIF: Eukaryotic initiation factor

ER: Endo plasmic reticulum

EMSA: Electrophoretic mobility shift assay

FHV: Flock house virus

xiv

g: Gram

GaMV: Galingsoga mosaic virus

GAPDH: Glyceraldehyde 3 - phosphate dehydrogenase

GDD: Gly - Asp - Asp domain gRNA: Genomic RNA

GTP: Gu anosine triphosphate

GRV: Groundnut rosette virus

H4: Hairpin 4

H4a: Hairpin 4a

H4b: Hairpin 4b

H5: Hairpin 5

HCRSV: Hibiscus chlorotic virus

HCV: Hepatitis C virus

HDV: Hepatitis delta virus

HIV: Human immunodeficiency virus

hpi: Hours post inoculation hr: Hour(s)

IGR: Intergenic region

JINRV: Japanese iris necrosis virus

K d : Dissociation constant

L: Liter

LB: Lysogeny broth

LS: Lower stem

xv

LSL: Large symmetrical internal loop

M1H: Motif 1 hairpin M3H: Motif 3 hairpin MDV: Qβ bacteriophage - associated midivariant RNA

mg: Milligram

min: Minute(s)

ml: Milliliter

mM: Millimolarity

MNSV: Melon necrotic spot virus

MP: Movement protein mRNA: Messenger RNA

MS salts: Murashige and Skoog basal salt mixture

N: Normality

ng: Nanogram

nt: Nucleotide(s)

OD: Optical density

ORF: Open reading frame

PABP: Poly(A) binding protein PCR: Polymerase chain reaction

PEG: Polyethylene glycol

PEMV: Pea enation mosaic virus

pH: Measure of acidity or alkalinity of a solution

PIM:

Protoplast isolation medium

xvi

pmol: Picomole

PMV: Panicum mosaic virus

PNK: Polynucleotide kinase

Pr: Core promoter for negative - strand initiation

PTGS: P ost transcriptional gene silencing

RCNMV: Red clover necrotic mosaic virus

RdRp: RNA - dependent RNA polymerase

RE: Replication enhancer

RNA: Ribonucleic

acid

rRNA: Ribosomal RNA

RT: Reverse transcription

SARS: Severe acute respiratory syndrome

satC: Satellite RNA C

satRNAs: Satellite RNAs

SDS: Sodium dodecyl sulfate

SELEX: Systematic Evolution of Ligands by Exponential Enrichment

SL: Stem loop

sgRNA: Sub genomic RNA STNV: Tobacco necrosis virus satellite RNA

TAV: T omato aspermy virus

TBSV: Tomato bushy stunt virus

TCV: Turnip crinkle virus

TGGE: Temperature - gradient gel electrophoresis

xvii

TMV: Tobacco mosaic virus

Tris: Tris[hydroxymethyl]aminomethane

T S S: T - shaped structure

TuMV: Turnip mosaic virus

TYMV: Turnip yellow mosaic virus

UTR: Untranslated region

UV: Ultraviolet

wt: Wild - type

Δ: Deletion

Ψ 1 : Pseudoknot 1

Ψ 2’: Pseudoknot 2’ Ψ 3 : Pseudoknot 3

Ψ 4 : Pseudoknot 4

α - 32 P: Alpha phosphorus - 32 γ - 32 P: Gamma phosphorus - 32 µ g: Microgram

µ L: Microliter

µ M : Micromolarity

µ mol: Micromole

1

CHAPTER I

ELEMENTS INVOLVED IN

REPLICATION OF POSIT IVE

STRAND RNA VIRUSES

Introduction

Positive strand RNA viruses comprise over one - third of known virus genera (van Regenmortel et al., 2000), and more than 75% of plant viruses.

Crop losses caused by (+) - strand RNA viruses have large economic influences worldwide. Positive strand RNA viruses, like P oliovirus , W est nile virus , and S evere acute respiratory syndrome coronavirus

(SARS), are major threats to

human s

and animals . To cont rol the diseases caused by (+) - strand RNA viruses, we need to understand the replication of these viruses.

Positive strand RNA viruses have a wide range of genome configurations. They can be either nonsegmented or segmented, have subgenomic RNAs, or can b e

associated with satellite RNAs ( satRNA )

or defective interfering RNAs ( DI RNA ) . Positive strand RNA viruses replicate through multiple steps involving the viral RNA - dependent RNA polymerase (RdRp), other viral proteins, and

the

host system

(Lai, 1998; Ma ckenzie, 2005) . As a (+) - strand RNA virus progresses through its life cycle, it takes various forms or conformations. Infection of a nimal viruses

is initiated by receptor - mediated endocytosis , whereas plant viruses are transmitted by insects or mechanical

abrasion . Upon infection, the virus uncoats from the virion and releases the viral (+) - strand RNA

2

into the cytoplasm. pH changes, membrane receptor binding, or protease activity may trigger this conformational change (Flint et al, 2004).

The viral RdRp is

then translated using the cellular translation machinery. At some point, translation stops and the same genomic RNA acts as the template for ( - ) - strand synthesis. Since translation (5 '

to 3 ' end) and transcription (3 '

to 5 '

end) cannot occur simultaneousl y , the initial viral (+) - strand RNA

needs to switch from

a translation - competent template to a replication - competent template

allowing ( - ) - strand RNAs to be transcribed. Newly synthesized ( - ) - strands then serve as templates for nascent (+) - strand RNA synthesis. Progeny (+) - strand

genomes have been

proposed to fold into a conformation that is not a ccessible to the RdRp, so the newly formed (+) - strand may not be a template for further ( - ) - strand synthesis (Zhang et al., 2006a; Zhang et al., 2006b). (+) - s trand and ( - ) - strand synthesis can be asymmetrical with up to 1000 (+) - strands synthesized for every one ( - ) - strand (Buck, 1996). Minus - strands also serve as templates for subgenomic RNA (sgRNA) synthesis

for viruses that have 3 '

coterminal subgenomic RNAs . sgRNAs, which code for structural and/or movement proteins, are produced by three possible mechanisms: premature termination of transcription by the RdRp during ( - ) - strand synthesis (Sit, Vaewhongs, and Lommel, 1998; Tatsuta et al., 2005; Wu et al., 2010 ); internal initiation on ( - ) - strands during (+) - strand synthesis

(Levis, Schlesinger, and Huang, 1990; Miller, Dreher, and Hall, 1985); or discontinuous transcription (Jeong and Makino, 1994).

In the life cycle of RNA viruses, packaging, or encapsidation

of the viral genome

by structural proteins is an essential step for transmission. Plant RNA viruses have different mechanisms for packaging. For monopartite Tymoviruses, the genomic RNA and sgRNA are packaged into separate virions. In the genus Luteovirus , only the genomic

3

RNA is packaged. The two RNA molecules from bipartite RNA viruses are packaged either separately into distinct virions (Comovirus and Nepovirus) or into the same virion (Dianthovirus). In the tripartite genera Bromovirus and Cucumovirus,

the largest two genomic RNAs are packaged individually into virions and the third genomic RNA and its sgRNA are co - packaged into a third virion. In the genus Alfamovirus, the three genomic RNAs and sgRNA are packaged individually into four distinct virions (Rao, 2006). sgRNA, satRNA, and DI RNAs are not required to initiate an infection, and

are

dependent on the parental virus for replication and packaging (Simon, Roossinck, and Havelda, 2004). Co - packaging of two or more RNA molecules requires either that

they all contain a packaging signal (Annamalai and Rao, 2005) or that RNAs without a packaging signal interact with a signal - containing RNA (Basnayake, Sit, and Lommel, 2006). Replication and transcription of (+) - strand RNA viruses are conducted by the Rd Rp. RdRp, along with the other three types of polymerases (DNA - dependent DNA polymerase, DNA - dependent RNA polymerase, RNA - dependent DNA polymerase), all have a right hand shape consisting of a palm, fingers and thumb domains, with the active site of the e nzyme located in the palm subdomain (Baker and Bell, 1998). In spite of their wide variation in genomic conformation and sequences, the four types of polymerases all have four common motifs. One critical motif is a highly conserved Gly - Asp - Asp (GDD) sequen ce located in the catalytic site of the enzyme (Jablonski and Morrow, 1995; Letzel, Mundt, and Gorbalenya, 2007; O'Reilly and Kao, 1998; Vázquez, Alonso, and Parra, 2000; Wang and Gillam, 2001; Wang et al., 2007).

4

Unlike DNA replication that requires a pr imer, RNA transcription by most (+) - strand RNA virus polymerases occurs de novo

(i.e., no primer needed), starting with the 3'

end. RdRp from some viruses can use DNA as a template to synthesize RNA, though it is about 10% as efficient as RNA synthesis wit h RNA templates (Siegel et al., 1999).

Viruses are considered to be molecular genetic parasites that utilize cellular systems for their own replication (Villarreal, 2005). They depend entirely on the host cells to reproduce their genome and form infectious

progeny. For all (+) - strand RNA viruses investigated so far, the viral replication complex co - purifies with cellular membrane extracts. Viral RNA synthesis takes place in virus - induced membrane compartments that are derived from different organelles

(Tabl e 1.1) , with viral proteins modifying the structure of intracellular membranes. Different viruses induce diverse but specific cellular structures, and can also use different membranes in the absence of the cognate organelle membrane. These virus - induced me mbrane structures often contain the viral replication complex and are thought to be the replication sites (Laliberté and Sanfaçon, 2010; Miller and Krijnse - Locker, 2008). The membranes provide a scaffold for anchoring the replication complex, which increas es the local concentration of components required for viral replication, and protects the viral RNA from being degraded by the host defense system. Although membrane association is important for (+) - strand RNA virus replication, the mechanism of replicatio n and the formation of these membrane structures are still poorly understood. In addition to viral replication proteins, cellular proteins have been found within virus - induced membrane structures including several proteins involved in protein synthesis an d folding. eIF(iso)4E, eEF1A, polyA - binding protein (PABP) and Hsp70 are

5

localized in vesicles induced by Turnip mosaic virus

(TuMV; Potyviridae ,

Potyvirus

genus) and are involved in viral RNA synthesis (Beauchemin, Boutet, and Laliberte, 2007; Beauchemin and Laliberte, 2007; Dufresne et al., 2008; Thivierge et al., 2008). Hsp70 and eEF1A are also found in Tomato bushy stunt virus

(TBSV; Tombusviridae , Tombusvirus

genus) - induced vesicles (Li et al., 2009; Wang, Stork, and Nagy, 2009). DnaJ and eIF3 are involved in Brome mosaic virus

(BMV; Bromoviridae , Bromovirus genus) RNA replication (Quadt et al., 1993; Tomita et al., 2003). The mechanism and function of these cellular proteins in viral RNA replication is not clear, with possibilities including stabilizin g viral replication proteins and controlling their activity.

6

Replication site

Family

Genus

Virus

Reference

ER membrane

Potyviridae

Potyvirus

Tobacco etch virus

(Schaad, Jensen, and Carrington, 1997)

Zucchini yellow mosaic virus

(Zechmann, Müller, and Zellnig, 2003)

Comoviridae

Nepovirus

Grapevine fanleaf virus

(Ritzenthaler et al., 2002)

Tomato ringspot virus

(Han and Sanfacon, 2003)

Cowpea mosaic virus

(Carette et al., 2000)

Alphaflexiviridae

Potexvirus

Potato virus X

(Bamunusinghe et al., 2009)

Bromoviridae

Bromovirus

Brome mosaic virus

(Restrepo - Hartwig and Ahlquist, 1996; Schwartz et al., 2002)

Tombusviridae

Dianthovirus

Red clover necrotic mosaic virus

(Turner et al., 2004)

unclassified

Tobamovirus

Tobacco mosaic virus

(Kawakami, Watanabe, and Beachy, 2004; Más and Beachy, 1999; Nishikiori et al., 2006; Reichel and Beachy, 1998)

mitochondrial membrane

Tombusviridae

Carmovirus

Melon necrotic spot virus

(Mochizuki et al., 2009)

Tombusvirus

Carnation Italian ringspot

(Weber - Lotfi et al., 2002)

peroxisomal membrane

Tombusviridae

Tombusvirus

Tomato bushy stunt virus

(McCartney et al., 2005b)

Cymbidium ringspot virus

(Navarro et al., 2006)

Cucumber necrosis virus

(Panavas et al., 2005; Russo, Di Franco, and Martelli, 1983)

chloroplast membrane

Potyviridae

Potyvirus

Turnip mosaic virus

(Prod'homme et al., 2003; Prod'homme et al., 2001; Wei et al., 2010)

Tymoviridae

Tymovirus

Turnip yellow mosaic virus

(Hatta, Bullivant, and Matthews, 1973),

Table 1.1 Cellular membranes associated with plant (+) - strand RNA viruses

7

Subviral RNAs Associated With Positive Strand RNA Viruses

Viruses can be associated with subviral RNAs (DI RNAs or satellite RNAs) that are dependent on the helper virus for replication, movement, and encapsidation (Simon, Roossinck, and Havelda, 2004). DI RNAs are derived mainly or completely from the genome of the helper virus, while by definition satRNAs have sequences either mostly or completely unrelated to any large contiguous segment of the helper virus genome. Plant RNA viruses are more frequently associated with

satellite RNAs. SatC from Turnip crinkle virus

(TCV; Tombusviridae , Carmo virus

genus) and B amboo mosaic virus

(BaMV; Flexiviridae, Potexvirus

genus) satRNA are among the be st studied. DI RNAs are commonly associated with animal RNA viruses, and have been found in a subset of plant viruses including carmoviruses, tombusviruses, bromoviruses, furoviruses, potexviruses, hordeiviruses, rhabdoviruses, and bunyavirus (reviewed by Simon and Bujarski, 1994). The best characterized plant virus DI RNAs are DI RNAs from TBSV and TCV.

O rigins of subviral RNAs

Animal and plant RNA viruses can form DI RNAs de novo

upon high multiplicity passage of DI RNA - free isolates, and are considered to be mistakes generated by the error - prone replicase during replication (Burgyan, Rubino, and Russo, 1991; Knorr et al., 1991; Li et al., 1989; Marsh et al., 1991; Resende et al.,

1991) .

Upon infection with in vitro

transcribed TCV (DI RNA - free), new low molecular weight RNAs accumulated that hybridized to TCV - specific probes. These new DI RNAs (e.g., DI1) were collinear

8

deletion mutants of TCV containing the exact TCV 5 '

and 3 '

en ds and an internal segment (Li et al., 1989). diG (346 nt), a DI RNA associated with TCV isolate TCV - B, is a mosaic molecule composed of 21 nucleotides of unknown origin at the 5 '

end, TCV 5 '

portion in the middle and TCV 3 '

terminus with a centrally repea ted block at the 3 ' end (Li et al., 1989). There are two different mechanisms proposed for DI RNA generation; enzyme cutting and ligation and replicase - driven copy choice (Simon and Bujarski, 1994). Recombination between TCV subviral RNAs support the repli case - driven copy choice model for the formation of DI RNAs (Cascone et al., 1990). During replication, when the replicase copies the viral ( - ) - strand, it can dissociate from the template prematurely. Before the newly made (+) - strand is released from the replicase complex, the replicase reinitiates (+) - strand synthesis either on the same template or on a different viral ( - ) - strand. Besides generation of DI RNAs, RNA recombination plays a pivotal role in virus evolution (reviewed by Simon and Bujarski, 1994). Since satRNAs share little sequence similarity with their helper viruses, their origin s remain

Full document contains 200 pages
Abstract: SatC is a noncoding subviral RNA associated with Turnip Crinkle Virus (TCV), a small (4054 nt) single-stranded (+)-strand RNA virus belonging to the Carmovirus genus. Because of its small size (356 nt) and TCV-derived 3' end, satC has been successfully used as a model to elucidate sequence and structural requirements for TCV RNA replication. Although satC is considered a model to identify cis-acting elements required for TCV replication, recent findings indicate distinct differences in structures and functions of these related sequences. RNA2D3D predicts that part of the TCV 3' end (H5, H4a, H4b and two pseudoknots) folds into an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits and is required for translation. SatC contains a similar 3' end with 6 nt differences in the 100 nt TSS region. RNA2D3D did not predict a similar structure for satC TSS region, and satC did not bind yeast ribosomes. satC nucleotides were changed into TCV TSS bases to determine which base differences are responsible for the loss of the TSS in satC. Changing these bases all increased ribosome binding but surprisingly none of them had an effect on satC accumulation in protoplasts and plants. Therefore satC may need these and other 3' end base differences for its required conformational switch for efficient replication, and not to inhibit ribosome binding. In vivo genetic selection (SELEX) of the linker sequence between H5 and the Pr showed the conservation of UCC, which led to the discovery of Ψ2 . Ψ2 is required for both viral and satC accumulation in protoplasts. H5-Pr linker had no significant structural change after RdRp binding in satC, which is different with TCV H5-Pr linker. TCV H5-Pr linker had a major structural change upon RdRp binding, and is proposed to be involved in a conformational switch. Replacement of satC H4a with randomized sequence and scoring for fitness in plants by SELEX resulted in winning sequences that contain an H4a-like stem-loop. SELEX of H4a/H4b in satC generated two different structures: wt H4a/H4b-like structure and a single hairpin structure. Two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. With the protection of CP, satC can form higher amount of dimers that have additional nucleotides at the junction sites in the absence of TCV. The extra nucleotides are not necessarily associated with an active TCV RdRp.