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

Little Cherry

Plant viruses cause many different symptoms

Vein-banding

Tissue Deformation

Necrosis

Flower Breaking

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.

Virions of plant viruses

Rigid rod

Icosahedral/ spherical

Flexuous rod

Plant virus genomes

Genes encoded by Tobacco mosaic virus

Tobacco etch virus genome

SP6 P1 HC-Pro P3 CI 6 NIa NIb Cap

TEV7DA-CMK
NcoI ClaI MluI KpnI NIa site (E N L Y F Q S) 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

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

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!)

Genes encoded by Tobacco mosaic virus

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

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

Replication of brome mosaic virus (BMV) in yeast: A system to discover host components involved in viral 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 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.

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 18:1 UFA/ increased 16:0 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

Spherules form the sites of viral replication in yeast, not well-developed in oleic acid mutants

Lee and Ahlquist. 2003. J. Virol. 77:12819-12828

General RNA virus replication strategy

Ahlquist et al. 2003. J. Virol. 77:8181-8186

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

Virus movement pathway

From Carrington et al. (1996) Plant Cell Vol. 8 (10):1669-1681

Steps in cell-to-cell movement

Cell-to-cell movement
1) 2) 3) Viruses need to leave sites of replication Viruses need to locate the plasmodesmata Viruses need to pass through plasmodesmata

Viral encoded movement proteins (MPs) facilitate these steps. Most MPs are Multifunctional.

Replication Movement to adjacent cells

1) 2) 3) 4) 5)

MPs are required for movement MPs bind to virus genomes MPs interact with plant cytoskeleton MPs localize to plasmodesmata MPs gate plasmodesmata

Plasmodesmata

Lucas. 2006. Virology. 344:169-184

Structure of plasmodesmata and comparison to viral particles

Genes encoded by Tobacco mosaic virus

Viral movement proteins

Lucas. 2006. Virology. 344:169-184

Model for trafficking through plasmodesmata

Lucas. 2006. Virology. 344:169-184

Role for cytoskeleton in TMV movement

Model for TMV movement

Lucas. 2006. Virology. 344:169-184

Model for Hordeivirus and Potexvirus movement

Lucas. 2006. Virology. 344:169-184

Another model for TMV movement that couples movement to replication

Kawakami et al. 2004. Proc. Natl. Acad. Sci. USA 101:6291-6

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

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. 77:12819-12828

Distribution of viral RNA at various stages after infection by TMV

Mas and Beachy. (1999) J. Cell Biology, 147 (5), 945958

Replicase and viral RNA colocalize early in infection

Mas and Beachy. (1999) J. Cell Biology, 147 (5), 945958