Plant Plus Strand RNA Viruses

SMALL RNA VIRUSES OF PLANTS

• Majority of plant viruses have genomes that consist of (+) sense single stranded RNA.

• A number of them have rod shaped helical morphology, while majority have icosohedral morphology.

• Infection by plant viruses differs from animal viruses because plant viruses are normally placed inside cells by vectors or during mechanical injury.

• Transmission in nature most commonly results from feeding of insect vectors such as aphids, leafhoppers, beetles, fungi and nematodes.

• No receptors have been identified for plant viruses.

Classification of Plant Viruses1)Most plant viruses have single

stranded RNA genomes & simple particle morphologies. The plus sense RNA viruses do not have envelopes and have monopartite, bipartite or tripartite genomes.

2)Rhabdovirus & Bunyavirus have negative strand RNA genomes, are enveloped are transmitted by insects and members of both families infect both plants and animals.

3) Ds RNA viruses belong to the Reovirus Family whose members encompass plants & animals.

4) Geminiviruses and Nanoviruses have ssDNA genomes with no close animal virus relatives.

5) The dsDNA viruses replicate via reverse transcription.

Plant Viruses Cause many different Symptoms

necrosis

Little Cherry

flower breakingTissue Deformation

Vein-banding

Rigid Rods Flexuous Rods Spherical Particles

Bacilliform Enveloped GeminatePleomorphic Enveloped

Some Plant Virus Particles

Basic Plant Virus Structures

Helix (rod) e.g., TMV

Icosahedron(sphere)e.g., BMV

Some Plant Viruses Induce Characteristic Inclusions

Potyvirus PinwheelsCitrus Tristeza InclusionsTMV Inclusions

Spherical Virus Inclusions CPMV Wall Inclusions CPMV MP in Wall

Experimental Mechanical Transmission of Plant Viruses

Agroinfection:

Vegetative Propagation of Plant Viruses

Many Viruses are Transmitted through Contact, Seed & Pollen

Plant Virus Life Cycle• Virus entry into host

– no attachment step with plant viruses– by vector, mechanical, etc. – must be forced– Requires a wound – delivery into cell

• Uncoating of viral nucleic acid– may be co-translational for + sense RNA viruses– poorly understood for many

• Replication– replication is a complex, multistep process– viruses encode their own replication enzymes

Plant Virus Life Cycle • Cell-to-cell movement

– cell-to-cell movement through plasmodesmata

– move as whole particles or as protein/nucleic acid complex (no coat protein required)

• Long distance movement in plant– through phloem– as particles or protein/nucleic acid complex

(coat protein required)• Transmission from plant to plant

– requires whole particles

Plant Virus Transmission

• Generally, viruses must enter plant through healable wounds - they do not enter through natural openings (no receptors)

• Insect vectors are most important means of natural spread

• Type of transmission or vector relationship determines epidemiology

• Seed transmission is relatively common, but specific for virus and plant

Plant Virus Transmission

• Mechanical transmission– Deliberate – rub-inoculation– Field – farm tools, etc.– Greenhouse – cutting tools, plant

handling– Some viruses transmitted only by

mechanical means, others cannot be transmitted mechanically

• Transmission by vectors: general– Arthropods most important– Most by insects with sucking mouthparts

• Aphids most important, and most studied• Leafhoppers next most important

– Some by insects with biting mouthparts– Nematodes are important vectors– Fungi may transmit soilborne viruses– Life cycle of vector and virus/vector

relationships determine virus epidemiology– A given virus species generally has only a

single type of vector

Plant Virus Transmission by Vectors

Arthropods as Virus Vectors

BeetlesWhitefliesThrips

Aphids Leafhoppers

• Carry Virus From Diseased to Healthy Plants.• Very Efficient Methods of Transmission & Movement.• Viruses & Arthropods have very Specific

Relationships. Mites

Nonpersistent Insect Transmission• Stylet-borne Viruses (Viruses Adsorb to Stylet)• Acquisition Period (Virus Acquired Immediately)• Latent Period (None)• Ability to Transmit Lost Quickly (Minutes to Hours)

Propogative Insect Transmission

• Viruses circulate in vectors and enter salivary Glands

• Acquisition period (Hours to days of feeding)• Latent Period (Hours to days after feeding)• Ability to transmit lost slowly (over many days)

Circulative Insect Transmission

• Viruses replicate in vectors• Acquisition period (Variable)• Latent Period (days after feeding)• Ability to transmit may last for lifetime• May be transmitted to progeny.

• Most use more than one strategy– Polyprotein processing– Subgenomic RNA– Segmented genome– Translational readthrough– Frameshift– Internal initiation of translation (without

scanning)– Scanning to alternative start site (truncated

product)– Alternative reading frame (gene-within-a-gene)

Plant virus genome expression strategies

Cell-to-Cell Movement • Plant viruses move cell-to-cell slowly

through plasmodesmata• Most plant viruses move cell-to-cell as

complexes of non-structural protein and genomic RNA

• The viral protein that facilitates movement is called the “movement protein” (MP)

• MPs act as host range determinants• MP alone causes expansion of normally

constricted plasmodesmata pores; MPs then traffic through rapidly

Plant cells are bound by rigid cell walls and are interconnectedby plasmodesmata, which are too small to allow passage ofwhole virus particles.

Plasmodesmata

Three different mechanisms have been described for cell-to-cell movement of plant viruses:

1. MP complexed with viral RNA moves along microtubules from ER-associated sites of viral replication; actin microfilaments deliver MP–RNA complexes to putative cell wall adhesion sites and plasmodesmata. These viruses do not require CP for cell-to-cell movement.2. NSP is a nuclear shuttle protein that moves newly replicated viral ssDNAgenomes from the nucleus to the cytoplasm. A movement protein, MPB, associated with ER-derived tubules, traps the NSP–ssDNAcomplexes in the cytoplasm and guides these along the tubules and through the cell wall into adjacent cells. These viruses also do not require CP for cell-to-cell movement.3. Some plant viruses move as whole particles through highly modified plasmodesmata (tubules). These viruses require CP in addition to MP for cell-to-cell movement.

Cell to Cell MovementPlants can restrict movement by hypersensitive resistance, and by resistant genes that form the plasmodesmata.Both Viral Genomes and Virions are known to move.

Systemic InfectionVirus moves through Phloem.Some viruses are restricted

to the phloem.Cells have 105 to 106 virions.The Meristem usually is free

of virus because of the lack of plasmodesmata.

Mosaics illustrate variationsof virus in leaf tissue.

Patterns of Systemic Movement of Viruses in Plants

TOBAMOVIRUS GROUPType member: Tobacco mosaic virus

Physical properties: Rigid rod shaped particles 18 X 300 nm, single infectious RNA, single coat protein

Transmission: Primarily mechanical

Major diseases: Severe mosaic of tobacco (TMV) and tomato (ToMV), mosaic of melons (cucumber green mottle mosaic virus, CGMMV), ringspot, mosaic and necrosis of orchids (odontoglossum ringspot virus, ORSV)

Tobacco Mosaic Virus– Important disease of tobacco, tomato & other plants.– Easily mechanically transmitted (only means of

transmission).– Very high concentration in plants.– First plant virus disease characterized (1898).– First virus strains demonstrated; – first cross protection shown.– First virus crystallized (1946 Stanley was awarded the

Nobel prize).– First demonstration of infectious RNA (1950s).– First virus to be shown to consist of RNA and protein.– First virus characterized by X-ray crystallography. – First plant virus genome to be completely sequenced.– First virus used for coat protein mediated protection. – First virus to have a resistance gene characterized.

PARTICLE STRUCTURE• Tobacco mosaic virus is best

studied example of a rod.• Each particle contains only a

single molecule of RNA (6395 nt) and 2130 copies of the coat protein subunit (158 aa; 17.3 kDa).– 3 nt/subunit– 16.33 subunits/turn– 49 subunits/3 turns

• TMV protein subunits + nucleic acid will self-assemble in vitro in an energy-independent fashion.

• Coat discs nucleate with TMV RNA at hairpin region.

• Self-assembly also occurs in the absence of RNA.

18 nm x 300 nm

Tobacco Mosaic Virus Symptoms

• Symptoms include mosaic, mottling, necrosis, stunting, leaf curling and yellowing of plant tissues. Some hosts develop resistance by forming necrotic lesions that constrain movement of the virus.

• Symptoms are very dependent on the host, the plant age, the environmental conditions, & the genetic background of the host plant and the virus strain.

GUSWT L252*-36

TMV local lesion assay

599-36

V73E-22 L252*-33V73E-1

Development of local lesions on the leaves of PAP mutant transgenic lines V73E-1, V73E-22, L252*-33 and L252*-36 upon infection with TMV. The untransformed tobacco and agus transgenic line were used as controls. The picture was taken 7 days post-inoculation.

TMV Causes Several Crop Diseases

Strains of TMV infect tomato and cause poor yield, distorted fruits, delayed fruit ripening and various fruit

discoloration problems that affect market values.

Tobacco mosaic virus is a typical positive-sense RNA virus with a 6.4 kilobase genome, has a cap at its 5’ end and a tRNA like structure at its 3’ end

Tobacco mosaic virus is a positive-sense RNA virus with a 5’ cap & a 3’ tRNA like structure.

6.4 KB GenomeEncodes Four ORFsTwo sg mRNAsAmounts of RdRp subunits regulated by translational readthrough. MP & CP are late proteins regulated by timing & amounts of mRNA synthesis. The MP mRNA isbicistronic with a silent CP cistron requiring expression from the monocistronic CP mRNA.

Three supergroups of positive strand RNA viruses

From Principles of Virology, Academic Press 1999

TMV Replication Cycle1)Virus enters injured cells.2) Ribosomes strip off coat and

begin to translate the RdRp.3) RdRp binds to the tRNA-like

3’ end & initiates synthesis ofminus strands to form RI

RNA. 4) Plus-strand genomic RNAs &

the movement protein (MP) & coat protein (CP) sgRNAs are synthesized.

5) MP & CP are translated.

6) MP forms complexes with newly synthesized viral RNA & membranes.The complex moves through plasmodesmata to adjacent cells.

7) CP & genomic RNAs accumulate to very high levels and virus crystals accumulate in the cytoplasm. Double-strand replicative form (RF) RNA accumulates in the cell and elicits host gene silencing responses.

8) Replication is very highly regulated but many details are not yet known.

virus particles

cell-to-cell movement

[54 kD]

MP (30 kD)

CP

transmission

RdRp

tRNAhis

mechanical

cap 126KMP

183K30K

17.5K

ribosomes drive co-translational disassemblyUAG*

mt/hel

CP

pppTranscription

tRNAhis

tRNAhis

ppp

cap

30K

17.5K54K

30K

17.5K

tRNAhis

[from –RNA]

CP

CP

CP

17.5K

vRNA

Rep

licat

ion

ER derived membranes

?

Translation126 kD, 183 kD

(RdRp)

Tobamovirus Multiplication

Cell-to-Cell Movement of TMV

1. Movement protein (MP) binds to TMV RNA to form MP complexes.2. Host proteins and/or other virus proteins may be in the MP-complex.3. The MP-complex moves from to adjacent cells through plasmodesmata,4. In the new cell, the viral RNA is released from the MP-complex. 5. The viral RNA is then translated on host ribosomes and the replication

cycle repeats.The Coat Protein Is Required for Vascular Movement!!!

Viral movement protein/viral RNA complex

MOVEMENT OF TMV IN THE INFECTED PLANT:• TMV uses its movement protein to spread from cell to cell through plasmodesmata.

• Normally, the plasmodesmata are too small for passage of intact TMV particles.

• The movement protein enlarges the plasmodesmatalopenings so that TMV RNA can move to the adjacent cells.

• TMV moves through plasmodesmata as a long, thin ribonucleoprotein complex composed of the TMV genomic RNA and the MP.

• As the virus moves from cell to cell, it reaches the plant’s vascular system for rapid systemic spread through the phloem to the roots and tips of the growing plant.

TMV-GFP:MP

Leading Edge

Late Uninfected

Confocal Microscopy Across Lesion(temporal relationships)

10 kD dextran move inward

10 kD dextrannot move

Time course of TMV GFP-MP effects on plasmodesmal gating and associations with the cytoskeleton (microtubules and actin filaments) and ER (aggregates) in different cells within an expanding lesion. Since virus is moving outwards from the center, cells at the leading edge are at early stages in infection while those at the center are at late stages in infection. Cartoon based on Heinlein et al. (1998) Plant Cell 10: 1107. Figure from Oparkaet al. (1996) Tr. Plant Sci. 1: 412.

Laarowitz (2001) in Fields Virology, 4th Ed. (Howley & Knipe, eds)

TMV MP Associations and Function in an Expanding Lesion

PDPD

ERER

PM anchoring sites

Nucleus cortical ER replication sites

microfilaments microfilaments

MPMP

sites of replication and protein synthesis

vRNAER derived membranes

TMV: A cytoplasmically replicating +RNA virus that moves without CP

Adapted from Lazarowitz (2001) in Fields Virology, 4th Ed. (Howley & Knipe, eds)

microtubulesmicrotubules

proteosome degradation, transport away from ER

TMV Assembly:Assembly initiates at the origin of assembly (OAS) sequence near the 3’ end.

Loop 1 of OAS is threaded through the center of a 2 layered disk made up of CP

The disk assumes lockwasher conformation and elongates in 3’to 5’ direction

Infected plants recover during Systemic Virus Invasion

GFP Engineered Plants GFP Virus Silencing

1) GFP Expression from virus vectors is very useful for monitoring gene silencing during virus invasion.

2 - Systemic movement patterns of viruses & GFP post transcriptional gene (PTGS) silencing are similar. Silencing signals inactivating transgenic GFP follow the same exit patterns from veins as virus expressing GFP.

3 - Viruses fail to invade meristems &PTGS is not active in meristems.

Dark Green Islands appearing during virus recovery lack virus. The light green areas contain large amounts of virus. The dark green islands and recovered tissue lack virus. The invaded leaf of the nontransgenic leaf has dark green islands that are resisting virus infection. The tip half of the transgenic leaf was invaded and the basal half exhibited recovery. Virus can not be detected in the dark green islands or in the recovered tissue. Recovery is based on gene silencing or RNAi.Non transgenic plant CP transgenic plant

Family Bromoviridae

Four bacilliform particles (30-57 X 18 nm) encapsidate:

gRNAs 1, 2, & 3 plus sgRNA4.

Bromoviruses: Brome Mosaic Virus (BMV)

Cucumoviruses:

Ilarviruses:

Alfamoviruses:

Tobacco Streak Virus (TSV)

Alfalfa Mosaic Virus (AlMV)

Cucumber mosaic virus (CMV)

graminaceous, legume plantstransmission: mechanical

solanaceous, legume plantstransmission: aphids

legume plantstransmission: aphids

woody plantsseed and pollen transmitted

Three IcosahedralParticles (26 to 35 nm) diameter Encapsidate four RNAs:

gRNA1, gRNA2, gRNA3 & sgRNA4

cap cap cap cap RNA 1 RNA 2 RNA 3 (sg)RNA 4

Replicase Replicase MP CPCP

Some Tripartite RNA Viruses

BROME MOSAIC VIRUS

• BMV particles are Icosahedra consisting of 180 coat protein subunits.

• Type member of the Bromovirus genus, family Bromoviridae.

• Virions are nonenveloped icosahedral (T=3), 26 nm in diameter, contain 22% nucleic acid and 78% protein.

• The BMV genome consists of three positive sense RNAs. RNA1 (3.2 kb) & RNA2 (2.9 kb), are encapsidated in separate particles. RNA3 (2.1 kb) & RNA4 (0.9 kb) are located in a third spherical particle.

RNA1 RNA2 RNA3RNA4

Divided RNA Genome of Brome Mosaic VirusViruses with divided genomes can efficiently express genes needed early in infection & can regulate the timing and amounts of late genes by synthesis of sgRNAs.

Brome mosaic virus is a tripartite RNA virus. Four proteins are expressed from three genomic RNAsRNA 1 encodes the helicasesubunit of the RDRP.RNA 2 encodes the polymerasesubunit of the RDRP.RNA 3 is bicistronic and encodes the movement protein and the coat protein.Ribosomes initiate at the 5’ m7G Cap of RNAs 1, 2 and 3 but can not initiate internally on RNA 3.RNA 4 is a sg mRNA translated from an internal promoter on the minus strand of RNA 3.

Brome Mosaic Virus RNAs

5’ m7G

tRNA-like

Polymerase Subunit

Movement

Helicase Subunit

Coat

3’

3’

3’

3’

5’ m7G

5’ m7G

5’ m7G

Coat

3.2 kB

2.9 kB

2.1 kB

0.9 kB

P

Permits Genome Reassortment in hosts infected with two viruses.

Molecular Genetic Analysis of Plus Strand RNA Viruses:

1) Transcription of synthetic RNAs from cDNA clones derived from viral genomes permit mutant analysis of specific biological traits and virus replication.

2) This strategy was first developed by Paul Ahlquist in 1984 with Brome Mosaic Virus. The method enabled analysis of Viral RNA for replication signals and promoters regulating mRNA transcription. In addition, application of foreign reporter genes provided numerous applications to assess replication.

3) Over the past 20 years Ahlquist’s strategy has been applied to most plus strand RNA viruses infecting plants, animals, and bacteria.

Mutate cDNA for specific changes & transcribe mutant Viral RNAs in vitro.

Inoculate mutant RNAs to host cells or protoplasts. Evaluate Biological Effects.

Viral RNA Genome

Reverse transcribe RNA & Clone cDNA.

Plasmid in bacteria to Amplify Cloned cDNA

Phage T7 promoter

BMV REPLICATION:• Proteins 1a and 2a are localized in the endoplasmic reticulum, the site of BMV RNA synthesis. ER-derived membranes are the sites of RNA synthesis for all (+) sense RNA viruses

• Promoter for the synthesis of (-) strand RNA is within the 134 nt tRNA-like sequence at the 3’ end of (+) sense genomic BMV RNAs

• RNA dependent RNA polymerase binds to this promoter sequence and initiates synthesis of (-) strand RNAs by a primer-independent mechanism from a 5’-CCA- 3’sequence

• A stem loop sequence within the tRNA-like domain and sequences upstream of the tRNA-like domain are required

Transcriptioncap CP tRNAtyr

sgRNA 4

mechanical

CP3acap tRNAtyrMP

RNA 3

2acap tRNAtyrGDD

RNA 2

1acap tRNAtyr

RNA 1

mt hel

CP (20 kD)

Translation

virus particles

RNA 1

RNA 3 +sgRNA 4

RNA 2

transmission

Translation

94 kD(RdRp)

109 kD 32 kD(MP)

Translation

cell-to-cell movement

?[or vRNAs?]

Bromovirus MultiplicationR

eplic

atio

n

vRNA 3

vRNA 1vRNA 2

Rep

licat

ion

From Lazarowitz (2001) in Fields' Virology 4th Ed. (Howley and Knipe, eds)

Subgenomic RNA synthesis requires the interaction ofreplicase with the promoter sequence in minus strand RNA3 that is directly upstream of RNA4 initiation sequence

Internal promoter for BMV subgenomic RNA synthesis

From Ishikawa et al. (1997) PNAS 94: 13810.

1a+2a directed RNA replication

X = URA3 (select for growth without uracil, or against growth in 5-fluoroorotic acid)

= CP, CAT, or GUS (assay for sgRNA synthesis and translation)

A Yeast System to Study BMV RNA Replication

1a and 2a expressed from 2µ plasmids (ADH promoter driver)

in vitro transcribed RNA 3 or by expressing RNA3 cDNA in vivo

5’-UTR and 3’-UTR sequences missing, so cannot replicateReplication is assayed by introducing RNA 3 either by introducing

Infection of yeast by Brome mosaic virus constructs

In the presence of RNA 1 and 2, the RNA3 transcript, which has its 5’-UTR and 3’-UTR, is correctly replicated via its complementary strand and subgenomic RNA4 is transcribed.

BMV REPLICATION IN YEAST

• RNA3 is correctly replicated in the presence of 1a and 2a in yeast.

• BMV 1a stabilizes RNA3 (+) sense RNA, increasing its half life from 5 min to 3 hours.

• The stabilization of RNA3 blocks its translation, 1a interacts with RNA3 to recruit it away from translation and into RNA replication.

• 1a interacts with the intercistronic 150 nt replication signal that includes a consensus box B-like element 5’-GGUUCAAyyCC-3’ found in RNA pol III promoters and in invariant residues of tRNA loops.

• Host proteins have been shown to be involved in stabilization of RNA3 through interactions with the 150nt element.