ANALYSIS OF GAG DOMAINS REQUIRED FOR RETROVIRUS ASSEMBLY

Project: Research project

Project Details

Description

Retroviruses are important etiological agents for human disease, most
notably causing acquired immunodeficiency syndrome (AIDS). Additional
human retroviral diseases are likely to be discovered, and these could
possess or acquire the ability to spread rapidly through the population.
At the present time, there are no cures or suitable vaccines to fight
retroviral diseases. Thus, it is necessary to learn as much as possible
about these viruses to provide inspiration for novel approaches to
control the diseases that they cause. One of the least understood parts
of their replication cycle is the process of retrovirion assembly. What
is clear is that the Gag proteins they encode play a central role.
Expression of the Gag protein results in the formation and release of
virus-like particles from the plasma membrane, even in the absence of all
the other components of the virion. The focus of this Continuation
Application is Gag-mediated particle assembly.

We have chosen Rous sarcoma virus (RSV) for our investigations because
it is one of the best understood retroviruses and has provided much of
what is already known about assembly. At the foundation of our proposed
experiments is a rapid and efficient transient expression system,
developed during the previous funding period, in which high levels of the
RSV Gag protein are made in mammalian cells. Particles are released from
the cells with full efficiency by budding from the plasma membrane and
contain all of the proper Gag cleavage products. Electron microscopy has
shown that the particles have an appearance that is indiscernible from
authentic RSV.

Using this system, we have recently obtained compelling evidence that
suggests that the RSV Gag protein can be diverted to the receptor for
pp60(src) (on the cytoplasmic face of the plasma membrane) for particle
formation. This finding implies that all of the functions required for
particle formation, except for membrane binding which also requires a
cell-encoded receptor, are self-contained within Gag itself. Experiments
are proposed to explore these ideas by placing appropriate signals on Gag
to direct it to other novel membrane sites for particle formation.

We have also completed an extensive deletion analysis of the RSV Gag
protein and found that surprisingly large sections of this protein can
be deleted without impairing the formation and release of particles.
Collectively, these non-essential regions amount to 503 of the 701 amino
acids in RSV Gag (72%). Moreover, our analysis has suggested the
existence of three discrete, movable domains that are essential for
particle formation. Defects in two of these domains can be complemented
in trans, but deletions in a third cannot, suggesting that it is the
region at which Gag-Gag interactions occur. Experiments are proposed to
thoroughly analyze the properties and functions of these assembly
domains, including their potential role in retroviral protease
regulation.
StatusFinished
Effective start/end date9/15/917/31/92

Funding

  • National Cancer Institute

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