Potyvirus Gene Expression and Genome Replication


Potyviruses are among the most numerous and important of plant virus families, with members that infect most known crops. These viruses are themselves members of the picorna superfamily of viruses, and share with picornaviruses characteristic genomic RNA structures, gene expression strategies, and presumably mechanisms of genome replication.

In our lab, we are using a range of approaches to understand in some detail the mechanisms that govern gene expressiona nd genome replication of tobacco vein mottling virus, or TVMV. One such approach is a transgenic approach. By expressing virus genes in plants, it is possible to interfere with the infection process, and in so doing gather information about processes vital for infection by TVMV. We have expressed each of the seven principle TVMV genes in tobacco, and found that the HC-pro (1), NIa (2), NIb (3), and CP (2) genes can all confer protection upon appropriately engineered transgenic plants. The characteristics of plants that express these genes are distinctly different, however. HC-pro and CP plants undergo a process known as recovery upon infection with TVMV; they develop a more-or-less normal infection, which abates in new leaves in the growing plant. CP plants differ from HC-pro plants in that CP plants are also protected against two other potyviruses (2), tobacco etch virus (TEV) and potato virus Y (PVY), whereas HC-pro plants are normally susceptible to other potyviruses (1).

In contrast, NIa and NIb plants rarely display symptoms upon inoculation with TVMV, and they are susceptible to other potyviruses (2,3). These contrasting characteristics have compelled us to examine whether the broad-range protection seen in CP plants could be combined with the strong resistance seen in NIa and NIb lines. Consequently, we have expressed the NIa, NIb, and CP genes as a single, self-processing polyprotein in tobacco. Plants that express this construction (with expression evaluated by examination of CP levels) are surprisingly sensiitive to TVMV and TEV. Thus, rather than addition of protection characteristics, these lines seem to have lost their resistance.

This observation maybe explained in a number of ways, and we have focused on one hypothesis for further study. We reason that the lack of protection in NIa-NIb-CP lines may be a consequence of association of the protein products of these genes with each other, instead of their "target" in infected cells. Thus, we have begun a survey of pairwise interactions between TVMV-encoded proteins using the two-hybrid system. We have documented interactions between the NIa, NIb, and coat proteins (4), and have obtained further genetic evidence that the NIb-CP interaction requires an intact "GDD" domain, and the NIa-NIb interaction a functional VPg domain.

More recent studies have focused on in vitro approaches. We have determined that the TVMV NIb gene product, purified from appropriately-programmed E. coli, possesses RNA polymerase activity (5). Our studies indicate that the highly-conserved GDD motif that is a hallmark of RNA-dependent RNA polymerases is important for the catalytic activity of NIb. These studies confirm that which had been presumed based on sequence homology - that potyvirus-encoded NIb proteins are in fact the virus-encoded RNA polymerase.

We have also established that the VPg and NIb proteins interact in vitro, by two criteria (6). First, immobilized GST-NIb is able to specifically retain VPg in a "pull-down" assay. Second, the VPg is able to stimulate, by some 5-fold, the polymerase activity of the NIb protein. These results lay a foundation for more extensive studies of the activity of the NIb protein. More generally, these developments opens new avenues of investigation into the mechanisms of potyvirus replication and pathogenicity.

Bibliography


1. Maiti, I. B., Von Lanken, C. D., and Hunt, A. G., unpublished observations

2. Maiti, I. B., Murphy, J. F., Shaw, J. G., and Hunt, A. G. (1993) Plants that express a potyvirus proteinase gene are resistant to virus infection. Proc. Nat. Acad. Sci. USA 90, 6110-6114.

3. Maiti, I. B., Von Lanken, C., Hong, Y., and Hunt, A. G. (1999) Introduction of multiple virus-derived resistance determinants into transgenic plants does not result in additive resistance properties. Journal of Plant Biochemistry and Biotechnology 8, 67-73.

4. Hong, Y., Levay, K., Murphy, J. F., Klein, P. G., Shaw, J. G., and Hunt, A. G. (1995) A potyvirus-encoded polymerase interacts with the viral coat protein and VPg in yeast cells. Virology 214, 159-166.

5. Hong, Y. and Hunt, A. G. (1996) RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase.Virology 226, 146-151.

6. Fellers, J., Hong, Y., Wan, J., Collins, G. B., and Hunt, A. G. (1998) In vitro interactions between a potyvirus-encoded genome-linked protein and RNA-dependent RNA polymerase. Journal of General Virology, 2043-2049.


Related papers from this lab


Domier, L. L., Franklin, K. M., Hunt, A. G., Rhoads, R. E., and Shaw, J. G. (1989). Infectious in vitro transcripts from cloned cDNA of the potyvirus, tobacco vein mottling virus. Proc. Nat. Acad. Sci. USA 86, 3509-3513.

Graybosch, R., Hellmann, G. M., Shaw, J. G., Rhoads, R. E., and Hunt, A. G. (1989). Expression of a potyvirus non-structural protein in transgenic tobacco. Biochem. Biophys. Res. Comm. 160, 425-432.

Berger, P., Hunt, A. G., Domier, L. L., Hellmann, G. M., Stram, Y., Thornbury, D., and Pirone, T. P. (1989). Expression in transgenic plants of a viral gene product which mediates insect transmission of potyviruses. Proc. Nat. Acad. Sci. USA 86, 8402-8406.

Murphy, J. F., Rhoads, R. E., Hunt, A. G., and Shaw, J. G. (1990). The VPg of tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24 kDa part of the proteinase. Virology 178, 285-288.

Murphy, J. F., Rychlik, W., Rhoads, R. E., Hunt, A. G., and Shaw, J. G. (1991). A tyrosine residue located within the small nuclear inclusion protein links TVMV VPg to its RNA. J. Virol. 65, 511-513.

Brantley, J. D. and Hunt, A. G. (1993). The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA binding protein. J. Gen. Virol. 74, 1157-1162.

Klein, P., Klein, R., Cerezo-Rodriguez, E., Hunt, A. G., and Shaw, J. G. (1994) Mutational analysis of the tobacco vein mottling potyvirus genome. Virology 204, 759-769.

Vance, V. B., Berger, P. H., Carrington, J. C., Hunt, A. G., and Shi, X. M. (1995) 5' proximal potyviral sequences mediate potato virusX/potyviral synergistic disease in transgenic tobacco. Virology 206, 583-590.

Murphy, J. F., Klein, P. G., Hunt, A. G., and Shaw, J. G. (1996) Replacement of the tyrosine residue that links a potyviral VPg to the viral RNA is lethal.Virology 220, 535-538.

Xu, D., Collins, G. B., Hunt, A. G., and Nielsen, M. T. (1997) Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene. Molecular Breeding 3, 291-306.

Xu, D., Collins, G. B., Hunt, A. G., and Nielsen, M. T. (1997) Factors affecting coat protein-mediated resistance against potyviruses in tobacco. Molecular Breeding 3, 331-339.

Fellers, J., Collins, G. B., and Hunt, A. G. (1998) The NIa-proteinase of different potyviruses provide specific resistance to virus infection. Crop Science 38, 1309-1319.

Xu, D., Collins, G. B., Hunt, A. G., and Nielsen, M. T. (1998) Resistance to alfalfa mosaic virus in transgenic burley tobacco expressing the AMV coat protein gene. Crop Science 38, 1661-1668.

Xu, D., Collins, G. B., Hunt, A. G., and Nielsen, M. T. (1999) Agronomic performance of transgenic burley tobaccos expressing the TVMV or AMV coat protein genes with or without virus challenges. Crop Science 39, 1195-1202.

Potyvirus alumni

  • Robert Graybosch
  • John Brantley
  • Peggy MacDonald
  • Indu Maiti
  • Isabel Tagavi
  • Yan Huang
  • Yiling Hong
  • John Fellers
  • Songhei Sen
  • Jinrong Wan
  • Also check out work from our collaborators:

  • John Shaw
  • Tom Pirone
  • John Murphy
  • Gary Hellmann
  • Vicki Vance
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