ANALYSIS OF RETROVIRUS ASSEMBLY BY IN VITRO MUTAGENESIS

Project: Research project

Project Details

Description

RNA tumor viruses are important etiological agents for human
disease, most notably causing human t-cell leukemia-lymphoma
and acquired immune deficiency syndrome (AIDS). In spite of
their importance, surprisingly little is known about the molecular
mechanisms by which the components of these viruses are
assembled. An understanding of this assembly process may be
essential to devising novel approaches for the control retroviral
diseases. The long-range goal of the work proposed here is to elucidate the
roles of the gag gene products in the assembly of Rous sarcoma
virus (RSV), the prototype retrovirus. The gag gene is known to
encode the sole viral products needed for budding of this avian
virus. These products do not function properly in mammalian
cells, and their functions are not well understood even in avian
cells. Attempts will be made to release the block to RSV replication
that occurs in mammalian cells by constructing a myristylation
site on the N-terminus of the gag product. The altered products
will be expressed using an SV40 vector. A cis-acting element of
gag that interferes with the replication of SV40-gag DNA will also
be located. To investigate RSV assembly in avian cells, an in depth study of
p27, the major capsid protein, will be undertaken. This gag
product is probably essential for assembly as well as infection.
The number, location, and function of the domains of p27 will be
sought using the method of suppressor-linker mutagenesis. This
method will allow the coding sequence of p27 to be saturated with
small insertions, and the effects of these mutations on virus
assembly will be determined after introducing them into avian
cells. Nonsense mutations will also be produced by the
mutagenesis scheme, and these will be used to determine the
minimal length of the gag product that will still permit budding.
For this purpose, a vaccinia virus vector will be developed to
express the wild-type and truncated gag products.
StatusFinished
Effective start/end date12/31/895/31/00