Download Plant Plus Strand RNA Viruses

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 Viruses
1)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.
Little Cherry
Plant Viruses
Cause many
different
Symptoms
Vein-banding
Tissue Deformation
necrosis
flower breaking
Some Plant Virus Particles
Rigid Rods
Flexuous Rods
Bacilliform Enveloped Pleomorphic Enveloped
Spherical Particles
Geminate
Basic Plant Virus Structures
Helix (rod)
e.g., TMV
Icosahedron
(sphere)
e.g., BMV
Some Plant Viruses Induce Characteristic Inclusions
TMV Inclusions
Citrus Tristeza Inclusions
Spherical Virus Inclusions
CPMV Wall Inclusions
Potyvirus Pinwheels
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
Plant Virus Transmission by Vectors
• 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
Arthropods as Virus Vectors
• Carry Virus From Diseased to Healthy Plants.
• Very Efficient Methods of Transmission & Movement.
• Viruses & Arthropods have very Specific
Relationships.
Aphids
Leafhoppers
Thrips
Whiteflies
Mites
Beetles
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)
Circulative 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)
Propogative 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.
Plant virus genome expression
strategies
• 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)
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 interconnected
by plasmodesmata, which are too small to allow passage of
whole 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 ssDNA
genomes from the nucleus to the cytoplasm.
A movement protein, MPB, associated with
ER-derived tubules, traps the NSP–ssDNA
complexes 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.
Patterns of Systemic Movement of Viruses in Plants
¾Cell to Cell Movement
Plants can restrict movement
by hypersensitive resistance,
and by resistant genes that
form the plasmodesmata.
¾ Both Viral Genomes and
Virions are known to move.
¾Systemic Infection
Virus 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 variations
of virus in leaf tissue.
TOBAMOVIRUS GROUP
Type 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 selfassemble 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.
TMV local lesion assay
WT
GUS
V73E-22
V73E-1
L252*-33
L252*-36
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 a
599-36
gus 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 Genome
Encodes Four ORFs
Two sg mRNAs
Amounts of RdRp subunits
regulated by translational
readthrough. MP & CP are
late proteins regulated by
timing & amounts of mRNA
synthesis. The MP mRNA is
bicistronic 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 Cycle
1)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 of
minus 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.
Tobamovirus Multiplication
mechanical
ribosomes drive co-translational disassembly
UAG*
MP
mt/hel
Translation
30K
126K
126 kD, 183
tRNAhis
183K
CP
(RdRp)
17.5K
RdRp
Replication
cap
Transcription
[from –RNA]
ppp
?
30K
CP
54K
ppp
tRNAhis
[54 kD]
tRNAhis
MP (30 kD)
tRNAhis
CP
17.5K
30K
CP
ER derived
membranes
17.5K
vRNA
cap
virus
particles
transmission
CP
17.5K
cell-to-cell
movement
kD
Cell-to-Cell Movement of TMV
Viral movement protein/
viral RNA complex
1.
2.
3.
4.
5.
Movement protein (MP) binds to TMV RNA to form MP complexes.
Host proteins and/or other virus proteins may be in the MP-complex.
The MP-complex moves from to adjacent cells through plasmodesmata,
In the new cell, the viral RNA is released from the MP-complex.
The viral RNA is then translated on host ribosomes and the replication
cycle repeats.
The Coat Protein Is Required for Vascular Movement!!!
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 plasmodesmatal
openings 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 MP Associations and Function in an
Expanding Lesion
Leading Edge
10 kD dextran move
inward
TMV-GFP:MP
Late
Uninfected
10 kD dextran
not move
Confocal Microscopy Across Lesion
(temporal relationships)
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 Oparka
et al. (1996) Tr. Plant Sci. 1: 412.
Laarowitz (2001) in Fields Virology, 4th Ed. (Howley & Knipe, eds)
TMV: A cytoplasmically replicating +RNA virus that moves
without CP
Nucleus
cortical ER replication sites
MP
PD
microfilaments
ER derived
membranes
vRNA
sites of replication and
protein synthesis
ER
microtubules
proteosome degradation,
transport away from ER
PM anchoring sites
Adapted from Lazarowitz (2001) in Fields Virology, 4th Ed. (Howley & Knipe, eds)
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
Non transgenic plant
CP transgenic plant
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.
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.
GFP Engineered Plants GFP Virus Silencing
3 - Viruses fail to invade meristems &
PTGS is not active in meristems.
Some Tripartite RNA Viruses
Bromoviruses: Brome Mosaic Virus (BMV)
graminaceous, legume plants
transmission: mechanical
Family Bromoviridae
Three Icosahedral
Cucumoviruses: Cucumber mosaic virus (CMV)
solanaceous, legume plants
transmission: aphids
Particles (26 to 35 nm)
diameter Encapsidate
four RNAs:
gRNA1, gRNA2,
gRNA3 & sgRNA4
Ilarviruses: Tobacco Streak Virus (TSV)
woody plants
seed and pollen transmitted
Alfamoviruses: Alfalfa Mosaic Virus (AlMV)
legume plants
Four bacilliform particles
transmission: aphids
(30-57 X 18 nm) encapsidate:
gRNAs 1, 2, & 3 plus sgRNA4.
RNA 1
cap
RNA 2
cap
Replicase
RNA 3
cap
Replicase
(sg)RNA 4
cap
MP
CP
CP
BROME MOSAIC VIRUS
RNA1
RNA2
RNA3
RNA4
• 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.
Divided RNA Genome of Brome Mosaic Virus
Viruses 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 RNAs
RNA 1 encodes the helicase
Brome Mosaic Virus RNAs
subunit of the RDRP.
tRNA-like
RNA 2 encodes the polymerase
Helicase Subunit
subunit of the RDRP.
5’ m7G
3’ 3.2 kB
RNA 3 is bicistronic and encodes
Polymerase Subunit
the movement protein and the
5’ m7G
3’ 2.9 kB
coat protein.
Ribosomes initiate at the 5’ m7G
Movement
Coat
Cap of RNAs 1, 2 and 3 but can not
2.1 kB
5’ m7G
3’
initiate internally on RNA 3.
P
RNA 4 is a sg mRNA translated
from an internal promoter on
the minus strand of RNA 3.
Coat
5’ m7G
3’
0.9 kB
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.
Viral RNA Genome
Phage T7
promoter
Reverse transcribe
RNA & Clone
cDNA.
Plasmid in bacteria to
Amplify Cloned cDNA
Mutate cDNA for
specific changes &
transcribe mutant
Viral RNAs in vitro.
Inoculate mutant RNAs to
host cells or protoplasts.
Evaluate Biological
Effects.
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
Bromovirus Multiplication
mechanical
RNA 1
1a
cap
mt
hel
tRNAtyr
109 kD
tRNAtyr
94 kD
(RdRp)
cap
RNA 2
cap
2a
GDD
Replication
Replication
RNA 3
Translation
Translation
3a
MP
CP
tRNAtyr
32 kD
(MP)
Transcription
cap CP tRNAtyr
sgRNA 4 Translation
CP (20 kD)
vRNA 3
RNA 1
vRNA 1
vRNA 2
RNA 2
RNA 3 +
sgRNA 4
virus particles
transmission
?
[or vRNAs?]
cell-to-cell
movement
From Lazarowitz (2001) in Fields' Virology 4th Ed. (Howley and Knipe, eds)
Internal promoter for BMV subgenomic RNA synthesis
Subgenomic RNA synthesis requires the interaction of
replicase with the promoter sequence in minus strand
RNA3 that is directly upstream of RNA4 initiation
sequence
A Yeast System to Study BMV RNA Replication
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)
1a and 2a expressed from 2µ plasmids (ADH promoter driver)
5’-UTR and 3’-UTR sequences missing, so cannot replicate
Replication is assayed by introducing RNA 3 either by introducing
in vitro transcribed RNA 3 or by expressing RNA3 cDNA in vivo
From Ishikawa et al. (1997) PNAS 94: 13810.
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.