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Robert Weldon, PhD
Research Interests:
The primary focus or my research laboratory is to understand host
cell contributions to retrovirus replication and in particular,
virus assembly and integration. Because a protective vaccine against
retroviral infections like HIV-1 has not yet been developed and
viruses resistant to current anti-retroviral therapeutics have
and will continue to emerge, there is an urgent need to search
for new antivirals to block viral replication. Clearly, a more
complete understanding of the molecular mechanisms underlying each
step of the retrovirus life cycle is an essential and compelling
platform for the discovery and development of new antiviral therapeutics.
Virus assembly and integration are two obvious targets for therapeutic
intervention and both are dependent upon the functions of the retroviral
Gag polyprotein. Gag proteins possess the ability to drive capsid
assembly and viral budding at the plasma membrane and it recruits
viral genomic RNA (vRNA) and other viral and cellular proteins
into the assembling virions. However, it is unclear where in the
cell Gag first recognizes the viral RNAs. Because Gag proteins
are thought to reside exclusively in the cytoplasm, the prevailing
hypothesis is that viral RNA recognition occurs as well. However,
recent data from our lab as well as others suggest that Gag proteins
from different retroviruses transiently associate with the nuclear
compartment through interactions with the nuclear shuttling components.
We have shown that the Mason Pfizer monkey virus (MPMV) Gag protein
interacts and colocalizes with the nuclear pore-associated, Sumo
conjugase Ubc9 in and around the nucleus. Several models are envisioned
which would explain these observations: 1) Ubc9 facilitates nuclear
import of a fraction of Gag proteins into the nucleus where vRNA
is first recognized and bound by Gag. The Gag-vRNA complex is then
exported to the cytoplasm where it initiates capsid assembly. 2)
Ubc9 directs Gag proteins to the nuclear pores where Gag proteins
accumulate and bind the vRNAs as they emerge through the nuclear
pores. These two models would explain how Gag proteins and ribosomes
compete for RNA packaging vs. translation. 3) Ubc9, through its
sumoylation activity, targets Gag to the plasma membrane. 4) The
trafficking activity of Ubc9 functions not during the late stages
of the virus life cycle but during the early stages to shuttle
the preintegration complex to the cellular chromatin via interactions
with mature Gag cleavage products. These models are not mutually
exclusive. Ubc9 may function during both the early and late stages
of the virus life cycle. We have now extended these studies and
found that Ubc9 also interacts with HIV Gag both in vitro and in
vivo. Thus, Ubc9 utilization appears to common to several retroviruses.
Our current goal is to determine the functional role(s) that Ubc9
plays during virus replication. For this, several strategies are
being used including mapping the Ubc9 binding site on both MPMV
and HIV-1 Gag proteins, utilizing RNAi technologies to knock down
Ubc9 expression and dominant negative mutants of Ubc9. These studies
will demonstrate whether Ubc9 is absolutely required for retrovirus
replication and identify which part of the virus life cycle this
protein is required.
The second focus of my laboratory is to identify the mechanism(s)
by which the retroviral capsid protein, Gag, is transported through
the cytoplasm to the plasma membrane. For this, we are using a
combination of real-time fluorescent confocal microscopy, FRET
(fluorescent resonance energy transfer) to document the precise
pathway of cytoplasmic protein transport and to screen for mutant
cell lines that are deficient for this obligatory step during viral
replication.
Recent Publications:
Weldon Jr., R. A., P. Sarkar, S. M. Brown, and S. K. Weldon. 2003.
Mason-Pfizer monkey virus Gag proteins interact with the human
sumo conjugation enzyme, Ubc9. Virology. 314: 62-73.
Weldon Jr., R. A., W. B. Parker, M. Sakalian, and E. Hunter. 1998.
Type D retrovirus capsid assembly and release are active events
requiring ATP. J. Virol. 72: 3098-3106.
Sakalian, M., S. D. Parker, R. A. Weldon, Jr., and E. Hunter.
1996. Synthesis and assembly of retrovirus Gag precursors into
immature capsids in vitro. J Virol. 70: 3706-3715.
Weldon Jr., R. A., and E. Hunter. 1996. Molecular requirements
for retrovirus assembly, p. 381-410. In W. Chiu, R. M. Burnett,
and R. L. Garcea (eds), Structural Biology of Viruses. Oxford University
Press, New York, NY.
Krishna, N. K., R. A. Weldon Jr., and J. W. Wills. 1996. Transport
and Processing of the Rous sarcoma virus Gag protein in the endoplasmic
reticulum. J Virology. 70: 1570-1579.
Weldon, R. A., Jr., and J. W. Wills. 1993. Characterization of
a small (25-kilodalton) derivative of the Rous sarcoma virus Gag
protein competent for particle release. J Virol. 67: 5550-5561.
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