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Virus host interactions

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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Virus host interactions

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  1. 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

  2. Plant viruses cause many different symptoms Little Cherry Vein-banding Flower Breaking Necrosis Tissue Deformation

  3. 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.

  4. Rigid rod Icosahedral/ spherical Flexuous rod Virions of plant viruses

  5. Plant virus genomes

  6. Genes encoded by Tobacco mosaic virus

  7. Tobacco etch virus genome SP6 P1 HC-Pro P3 CI 6 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

  8. 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

  9. 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 (doesn’t have to be particles!)

  10. Genes encoded by Tobacco mosaic virus

  11. 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

  12. 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), 945–958

  13. 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 transcribedRNA 3 originally transfected in yeast From Ishikawa et al. (1997) PNAS 94: 13810.

  14. 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. 2097–2106

  15. 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

  16. General RNA virus replication strategy Ahlquist et al. 2003. J. Virol. 77:8181-8186

  17. 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

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

  19. Steps in cell-to-cell movement • Viruses need to leave sites of replication • Viruses need to locate the plasmodesmata • 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

  20. Plasmodesmata Lucas. 2006. Virology. 344:169-184

  21. Structure of plasmodesmata and comparison to viral particles

  22. Genes encoded by Tobacco mosaic virus

  23. Viral movement proteins Lucas. 2006. Virology. 344:169-184

  24. Model for trafficking through plasmodesmata Lucas. 2006. Virology. 344:169-184

  25. Role for cytoskeleton in TMV movement

  26. Model for TMV movement Lucas. 2006. Virology. 344:169-184

  27. Model for Hordeivirus and Potexvirus movement Lucas. 2006. Virology. 344:169-184

  28. Another model for TMV movement that couples movement to replication Kawakami et al. 2004. Proc. Natl. Acad. Sci. USA 101:6291-6

  29. 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

  30. 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

  31. Distribution of viral RNA at various stages after infection by TMV Mas and Beachy. (1999) J. Cell Biology, 147 (5), 945–958

  32. Replicase and viral RNA colocalize early in infection Mas and Beachy. (1999) J. Cell Biology, 147 (5), 945–958

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