Virus host interactions

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Virus host interactions

• Strategies viruses use to replicate their genomes
  in
• susceptible host cells replication
• Strategies viruses use to move their genomes
  throughout
• susceptible host plants cell-to-cell
  movement
• -Strategies viruses use to suppress host defenses

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Plant viruses cause many different symptoms
Little Cherry
Tissue Deformation
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Symptoms of TMV in Tomato
Strains of TMV infect tomato and cause poor
yield, distorted fruits, delayed fruit ripening
and various fruit discoloration problems that
affect market values.
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Virions of plant viruses
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Plant virus genomes
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Genes encoded by Tobacco mosaic virus
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Tobacco etch virus genome
SP6
HC-Pro
P1
P3
CI
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NIa
NIb
Cap
TEV7DA-CMK
(E N L Y F Q S)
NcoI
ClaI
MluI
KpnI
NIa site
bar GFP GUS
TEV-bar TEV-GFP TEV-GUS
GUS
GFP
bar
GUS beta-glucuronidase (turns colorless
substrate to blue precipitate), quantitative GFP
green fluorescent protein (fluoresces green
under ultra violet light), non-destructive bar
basta resistance (herbicides containing basta or
glufosinate), easy selection
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Viral Pathogenesis
Within the plant, viruses must complete three
major steps in order to infect a susceptible
plant host.
Infection of single cells
Cell-to-cell movement
Long-distance movement

• Replication

• Replication
• Movement to
• adjacent cells

• Replication
• Movement to
• adjacent cells
• Movement to
• cells throughout
• the plant

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General features of viral replication
Infection of single cells

• Steps involved in positive-stranded
• RNA virus replication
• 1) Virus enters cells and 2) is uncoated
• 3) Viral genomic RNA is translated to
• produce replicase proteins
• 4) (-)-strand synthesis
• 5) ()-strand synthesis of sub-genomic RNAs
• 6) Synthesis of viral proteins
• 7) Assembly into virions
• 8) Movement as ribonucleoprotein complexes
• (doesnt have to be particles!)

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Genes encoded by Tobacco mosaic virus
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Role of the host in replication
Host proteins and structures are associated with
sites of viral replication and with replicase
proteins
Basic strategies used so far

• Cell Biology
• Biochemistry
• Forward and reverse genetics in plants and
  heterologous organisms,
• such as yeast

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Viral RNA colocalizes with BiP marker for
endoplasmic reticulum (ER) TMV replicates in
association with the endoplasmic reticulum
Mas and Beachy. (1999) J. Cell Biology, 147 (5),
945958
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Replication of brome mosaic virus (BMV) in yeast
A system to discover host components involved in
viral replication
1a2a 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 2m plasmids (ADH
promoter driver)
5-UTR and 3-UTR sequences missing, so cannot
replicate
in vitro transcribed RNA 3 originally transfected
in yeast
From Ishikawa et al. (1997) PNAS 94 13810.
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Composition of fatty acids is critical
for replication of brome mosaic virus in yeast
cells-genetic support for importance of host
membranes in viral replication
BMV RNA3-GUS and -CAT are not replicated in oleic
acid deficient yeast (decreased 181
UFA/ increased 160 SFA)
BMV replication is restored by complementing
ole1 mutation. Genetic proof of requirement for
oleic acid.
Lee et al. JOURNAL OF VIROLOGY, Mar. 2001, p.
20972106
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Spherules form the sites of viral replication in
yeast, not well-developed in oleic acid mutants
Lee and Ahlquist. 2003. J. Virol. 7712819-12828
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General RNA virus replication strategy
Ahlquist et al. 2003. J. Virol. 778181-8186
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Virus movement
After the virus replicates it has to be able to
move to new cells and new tissues/organs in order
to systemically infect
Infection of single cells
Cell-to-cell movement
Long-distance movement

• Replication

• Replication
• Movement to
• adjacent cells

• Replication
• Movement to
• adjacent cells
• Movement to
• cells throughout
• the plant

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Virus movement pathway
From Carrington et al. (1996) Plant Cell Vol. 8
(10)1669-1681
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Steps in cell-to-cell movement

1. Viruses need to leave sites of replication
2. Viruses need to locate the plasmodesmata
3. Viruses need to pass through plasmodesmata

Cell-to-cell movement

• Viral encoded movement proteins (MPs)
• facilitate these steps. Most MPs are
• Multifunctional.
• MPs are required for movement
• MPs bind to virus genomes
• MPs interact with plant cytoskeleton
• MPs localize to plasmodesmata
• MPs gate plasmodesmata

• Replication
• Movement to
• adjacent cells

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Plasmodesmata
Lucas. 2006. Virology. 344169-184
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Structure of plasmodesmata and comparison
to viral particles
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Genes encoded by Tobacco mosaic virus
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Viral movement proteins
Lucas. 2006. Virology. 344169-184
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Model for trafficking through plasmodesmata
Lucas. 2006. Virology. 344169-184
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Role for cytoskeleton in TMV movement
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Model for TMV movement
Lucas. 2006. Virology. 344169-184
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Model for Hordeivirus and Potexvirus movement
Lucas. 2006. Virology. 344169-184
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Another model for TMV movement that couples
movement to replication
Kawakami et al. 2004. Proc. Natl. Acad. Sci. USA
1016291-6
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Summary

• - Plant viruses encode proteins that direct the
  replication and movement
• of their genomes
• Viral replication occurs in association with
  host membranes and host factors
• Viral movement is directed by movement proteins
  that serve many functions
• binding the viral genome
• transporting the viral genome to plasmodesmata
• gating plasmodesmata
• trafficking through plasmodesmata

• Viral movement proteins interact with host
  proteins to accomplish their functions
• cytoskeleton
• kinases
• chaperones
• docking proteins

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BMV 1a replicase protein localizes to the
appropriate subcellular membranes, but does not
induce spherule formation in oleic acid deficient
mutants
Lee and Ahlquist. 2003. J. Virol. 7712819-12828
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Distribution of viral RNA at various stages
after infection by TMV
Mas and Beachy. (1999) J. Cell Biology, 147 (5),
945958
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Replicase and viral RNA colocalize early in
infection
Mas and Beachy. (1999) J. Cell Biology, 147 (5),
945958