UNIT I

PLANT-VIRUS INTERACTIONS

The Bible of Plant Virology

Hull, R. 2014. Plant Virology. 5th Ed.

Academic Press. London. UK.
 PLANT VIRUSES:
INFECTION AND REPLICATION
 WHAT IS A VIRUS?
A Virus may be defined as an
acellular biological entity with a
nucleic acid genome, and which
can direct its own replication
inside host cells using the
metabolic machinery of the host.

Viruses differ from cells in three fundamental ways:

A virus usually has only a single type of nucleic acid serving as
its genetic material.
Viruses contain no metabolic enzymes required for generation
of energy storing compounds such as ATP.
 Viruses do not encode the enzymatic machinery to synthesize
their proteins. Thus, they are dependent on host cells for these
processes.
Viruses replicate by
assembly of preformed
components into particles
No growth or binary
fissions
Simple viruses are composed of nucleoprotein.
The protein provides a protective coat.
The Viral Genome consists of either DNA or RNA.
Complex viruses may have other components,
including Carbohydrates, Lipids & Phosphoproteins.

Complex viruses may also have

viral encoded enzymes.
David Baltimore’s Viral Genome Classification
Scheme
Group VI
 Plant virus infection is always systemic

TMV-induced nll in tobacco

with resistance gene N

TMV infection in tobacco cv. Samsun

Host colonisation by plant viruses

Samuel. 1934. Ann. Appl. Biol. 21:90

 Plant-Virus Interactions

• Susceptible interactions
– Translation
– Replication
– Movement
• Resistance responses
– RNA silencing - VIGS
– Dominant resistance – active effect
– Recessive resistance – passive effect
Counting necrotic local lesions
allows virus quantification
Virus labelling with fluorescent proteins

Zwart et al. 2011. PLoS Pathogens 7:e1002122

Relationship between inoculum concentration and
number of infection foci
Independent action hypothesis

- Each virion has a certain

probability of infection
- During infection virions act
independently from each other

Zwart et al. 2011. PLoS Pathogens 7:e1002122

TMV replication in tobacco leaves

Goodchild et al. 1958. Virology 5:561

Penetration through the cell wall
 Expression of Plus-strand RNA Virus Genomes

 Eukaryotic plus strand RNA viruses replicate in the

cytoplasm.
 Cells do not express the enzymes required to replicate or
transcribe viral RNAs.
 Virus must provide its own RNA-dependent RNA
polymerase (RdRP) for replication and transcription.
Viral genome is both mRNA for gene expression and
template for replication.

 Viral mRNAs must follow the rules of their host cells.

 Eukaryotic RNA viruses must adapt to monocistronic
translation strategies of eukaryotic mRNAs.
Central problem for RNA viruses:
Hosts do not encode RNA-dependent RNA-polymerases.
Must follow rules for translation of eukaryotic mRNAs.
Central problem for DNA viruses:
Differentiated, quiescent cells do not support replication
of DNA.

Induction of host replication enzymes and cell cycle

regulators.
 Replication always requires expression of at least one
(or many) virus protein
 Host provides the rest of necessary protein, and at least
one.
Replication initiates at defined sites called origins
Replication initiation always requires a primer.
Disassembly and translation in vivo

Wilson (1984)
Infection and Replication

Virus Disassembly
(Co-translational)
 Translation

40S

60S

Translation
Factors
 Translation

40S
O

60S
 Translation

40S

60S
 Replication

RdRp
 Replication

RdRp
 Replication

RdRp
Movement complexes
Movement complexes
Virus Particles (Virions)
ss (+)-strand RNA Virus Genomes have three functions

Nicholson & White (2014). Nat Rev Microbiol.

 GENE EXPRESSION AND REPLICATION
ss (+) RNA VIRUSES

Small genomes (3.5- 32 kb) that maximise their coding

capacity according to different strategies
 Diversity of plant (+)ssRNA virus genomes
Genome (kb)

Closterovirus 13-19

Potyvirus 9.7

Tobamovirus 6.4

Polerovirus 5.9

Carmovirus 4.0
 Diversity of plant (+)ssRNA virus genomes
Genome (kb)

Comovirus 9-10

Bromovirus 8

Benyvirus 14-16
Assembly of the translation machinery

Robaglia & Caranta 2006. Trends Plant Sci. 11:40

Non-canonical initiation of translation
Non-canonical initiation of translations: Internal Ribosom
Entry Site (IRES)

Johnson et al. 2017. Proc R Soc B. 372: 20160177

Role of host factors in translation of (+)ssRNA viruses

Hyodo & Okuno. 2016. Curr. Op. Virol.. 17:11

 Non-canonical elongation

Virus infection subverts the protein synthesis machinery of the host cell

Walsh & Mohr. 2011. Nature Rev. Microbiol. 9:860.

Barley Yellow Dwarf Virus: a good example of complexity
in viral expression strategies
Summary of BYDV Translational Regulation

 Cap independent
translation.
 RdRP frameshifting.

 Cap-Independent translatio
 Leaky scanning.
 Translational readthrough

No Protein expression??

Miller & Rasochová (1997) Ann Rev Phytopath 35: 167-190

 MNSV-Mα5 vs 264: Virulence in melon and in N.
benthamiana

SL A

MNSV-264
SL B

MNSV SL A
avirulent
2

2 SL B

5 5

3
3
Melon S
Melon R
N. benthamiana

SL C
SL C
Truniger et al., (2008) Plant Journal 48, 452-462
 MNSV-Mα5 vs 264: Virulence and translational competence
correlate

120
Susceptible melon
100

80

60

40 5´-UTR 3´-UTR
LUC
20

0
T7-luc 3´-UTR-Ma5
3´-plasmid 3´a5 3´264
3´-UTR-264 Q3*SL
3´-Ma5+264 Q3SL
3´-264+Ma5 120

160
N. benthamiana
Resistant melon 100
140

120 80

100
60
80

60 40

40
20
20
0
0
1 2 3 4 5
T7-luc 3´-UTR-Ma5
3´-plasmid 3´a5 3´264
3´-UTR-264 Q3*SL
3´-Ma5+264 Q3SL
3´-264+Ma5 3´-plasmid 3´-UTR-Ma5 3´-UTR-264 3´-Ma5+264 3´-264+Ma5
MNSV-Mα5: a 5´-3´-UTR interaction needed for efficient
translation
SL3
SL2
40 60-
5´-UTR MNSV-Mα5

30

20- -80

SL1

10

3´-UTR SLC MNSV-Mα5 3´-UTR SLC MNSV-264

MNSV-Mα5: a 5´-3´-UTR interaction needed for efficient
translation
 MNSV-Mα5: eIF4E dependence
eIF4E-S complements MNSV-Mα5 in N. benthamiana

Nieto et al., 2011, Plant Journal 66:492-501

 Replication of (+) ssRNA viruses occurs in the cytoplasm

2. CP is stripped from RNA 3. Host ribosomes translate RdRP

1. Entry into
host cell 4. Minus sense
RNAs synthesized
as templates for
synthesis of plus
sense RNAs

7. Progeny
genome are
coated to form
5. Plus sense
new particles
sgRNAs are
(up to106 to
synthesized for
107 Virions/cell)
expression of
other viral genes

6. Infection progresses to other host cells

 Polarity of RNA transcription is 5’ to 3’

t(0) 5’
RdRP = RNA-dependent RNA polymerase

5’

t(1) 5’ 3’

5’

t(2) 5’ 3’

Growth of the nascent chain is from the 3’ end, i.e., 5’ to 3’

synthesis of the growing chain.
Movement of the transcription complex on the template is 3’ to 5’,
because of the antiparallel relationship between the two RNA
strands.
 Replication is not semi-conservative
5’+ 5’+
- 3’ 5’ +
*-
5’ -* +
Synthesis of Synthesis of
Synthesis of Minus-Strand Plus-Strand
Intermediates Progeny RNAs
*
Minus-Strand *
By RdRp By RdRp
or *
*
*
*
- 5’- 5’
+
5’
+
5’

3’ 3’ 5’ 3’ 5’ - 5’ 3’ 5’
+
+5’
Genomic RNA Replicative Replicative Replicative
(+ssRNA) Form Intermediate I Intermediate II

Intermediates are double-stranded RNAs; Asymetrical copying of RNA

intermediates occurs.
No proof reading is available: replication highly error prone (10-4 – 10-4/
base & round).
Most RdRP’s Recognize Structures at the 3’ Ends of Viral RNAs
 ViralRNAs have highly base-paired structures at their 3’ ends,
organised in stem-loops and pseudoknots.
 RdRp’s are able to discrimimate between viral and cellular RNAs by
recognizing these termini.
TMV 3’ Structure

RNA Folding

BMV RNA2 5’ Structure BMV RNA 3’ Structure

Artist’s Rendition Recruiting Element Promoter for (-)strand

synthesis
Structure of RdRps

A
D E

B
C

Poliovirus RdRp
 Eukaryotic RNA virus replication

Problem: The replicases of eukaryotic viruses are

membrane-bound.
Attempts to solubilise viral replicases from host
membranes result in reductions in enzymatic activity
and in template specificity.
 TMV Replication Complexes Are Associated With
Membranes

Membranes and RdRp Enlarged View

Confocal microscopy of protoplasts 24 hr post inoculation.

Thanks to Roger Beachy
 Brome mosaic virus (BMV) as a model
for ssRNA virus replication

1a 2a 3a 3b

CP

Brome Mosaic Virus Genome Organization and Function

 Two RdRP subunits are virus-coded. The 1a protein contains methyl transferase (MT)
and helicase motifs and the 2a protein contains the polymerase (GDD) motif.
 The 1a protein stabilizes BMV genomic RNAs, blocks their translation and causes
formation of membrane vesicles in host cells.
 The 1a associates with membrane bodies and interacts with the 2a subunit. The
helicase & MT domains are required for formation of 1a:RNA complexes.
The 2a protein interactions require N-terminal amino acids and the polymerase
domain. .
BMV RNA Replication can be studied in a Yeast System

1a+2a directed RNA

replication

X = URA3 (select for growth without uracil, or against

growth in 5-fluoroorotic acid).
Or, X = CP, CAT, or GUS (assay for sgRNA synthesis and
translation).

BMV 1a and 2a protein are expressed from yeast plasmids from an inducible
promoter (GAL1).
The authentic 5’-UTR and 3’-UTR sequences are deleted; therefore BMV RNA 1 and
2 do NOT replicate.
In vitro transcribed RNA 3 can replicate and produce sgRNA for expression of MP
and CP (or CP-substituted marker gene).

Yeast is a powerful tool for identification of host factors and analysis of

replication complexes.

Ishikawa et al. 1997. PNAS 94: 13810.

Electron Micrograph of Yeast Cells Expressing:
Cell Wall (CW)
No BMV Plasma Membrane (PM)
Nucleoplasm (Nuc);
Cytoplasm (Cyto)
Vacuole (V)
1a Protein
BMV 1a protein induces
perinuclear, ER membrane
spherules (vesicles) in yeast.

RNA 3 is also associated with the

vesicles (Not Shown).
1a &2a Proteins
+RNA3 ER lumen (ERL). Arrowheads
indicate splitting/rejoining of the
Schwartz et al outer and inner nuclear
Molecular Cell envelope
9:505 (2002).

The spherules are the sites of viral replication.

 Genome Wide Screening to identify Yeast Genes Affecting
BMV Replication

RNA and Western blot assays for BMV RNAs and proteins from wild type
yeast and yeast deletion strains with reduced BMV-directed replication. Nearly
100 genes were identified.

Kushner et al., PNAS 100:15764 (2003).

BMV Replicates in Vesicles Induced by the 1a RdRP Protein

The spherules are the sites of negative strand and positive

strand viral RNA synthesis

Díaz & Wang. 2014. Curr. Op. Virol. 9:104

Role of host factors in biogenesis fo VRCs

Hyodo & Okuno. 2016. Curr. Op. Virol.. 17:11

Virus re-arrangements of host membranes

Laliberté & Zheng. 2014. Annu. Rev. Virol. 1:237

 References

Firth & Brierly 2012. Non-canonical translation in RNA viruses. J. Gen. Virol.
93: 1385

Walsh & Mohr. 2011. Viral subversion of the host protein synthesis machinery.
Nature Rev. Microbiol. 9:860

Nagy & Pogany. 2012. The dependence of viral replication on co-opted host
factors. Nat. Rev. Microbiol. 10, 137

Laliberté & Zheng. 2014. Viral manipulation of plant host membranes.

Annu. Rev. Virol. 1:237

Hyodo & Okuno. 2016. Pathogenesis mediated by proviral host factors involved in
translation and replication of plant positive-strand RNA viruses.
Curr. Op. Virol. 17.:11