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.
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.
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
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
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
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,
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.
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