BIOCHEMICAL AND GENETIC STUDIES OF MULV ENV PROTEINS

Project Details

Description

The murine leukemia virus (MuLV) env gene is an important
determinant of viral infectivity, and the products of this gene
provide useful models for studying the biosynthetic pathways of
membrane proteins. The spleen focus-forming virus (SFFV), a
defective MuLV variant, is a potent pathogenic agent, causing
extensive proliferation of spleen cells shortly after infection and
leading to the rapid development of erythroleukemia. Although SFFV
contains a defective env gene which is not involved in viral
replication, genetic evidence strongly implicates the SFFV env gene
as the major pathogenic determinant of these viruses. We have
recently found that several SFFV glycoproteins, including the
mature env gene product, gp65, are efficiently secreted from cells,
and that partially purified preparations of gp65 stimulate the
proliferation of erythroid progenitor cells in vitro. These
results suggest that gp65 or a related component may be the
molecule responsible for the pathogenicity of these viruses. We
now propose to extend our studies of both the MuLV and SFFV env
components, to further elucidate the structure and function of
these components and to clarify their roles in the leukemogenic
process. The functional roles of oligosaccharide substituents of gp70 in the
transport and secretion of the MuLV env proteins will be determined
by treatment with a number of drugs which interfere with the normal
processing of N-linked sugars. The structures of the
oligosaccharides on molecules secreted in the presence of these
drugs will be determined, and the assembly and infectivity of the
resulting virions analyzed. The sites of O-glycosylation on gp70
will be identified, and the nature of the O-linked sugars
determined. Extracellular SFFV products will be purified to
homogeneity and the proliferative activities of the purified
material for hematopoietic cells will be assayed. Further
biochemical studies of SFFV glycoproteins will be preformed,
including amino- and carboxy-terminal sequencing and the complete
characterization of the post-translational modifications of these
molecules. Site-specific mutations will be introduced into
molecularly cloned genomes of SFFV and MuLVs by oligonucleotide-
directed mutagenesis, in order to determine the functional roles
of O-linked glycosylation and the structural features specific for
the SFFV env components in the biological activities of these
proteins and in the pathogenicity of these viruses. Monoclonal
antibodies and site-specific antisera will be prepared against
different domains of gp65 and the active site of gp65 identified
by examining the effects of such antibodies on the in vitro and in
vivo biological activities of this molecule. The effect of the
regulatory Fv-2 gene on the response of hematopoietic cells to gp65
will be examined as an additional test of the biological
specificity of the proliferation activity of this molecule.
Finally, binding assays will be developed for both gp70 and gp65,
and cell surface receptors for these molecules isolated and
characterized in order to study the role of receptor interactions
in viral leukemogenesis.
StatusFinished
Effective start/end date12/31/8912/31/92

Funding

  • National Cancer Institute

ASJC

  • Genetics
  • Medicine(all)
  • Biochemistry

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