| Abstract |
The aim of this proposal is to isolate
the potent intracellular HIV-1 inhibitor, termed Lv1, which
has been recently described in the cells of Rhesus macaques
and some other nonhuman primates. We propose a novel genetic
approach for the isolation of Lv1 using a Rhesus genomic cosmid/episomal
library as a source of Lv1. The advantages to using Rhesus
genomic DNA, rather than a Rhesus cDNA expression library
as a source for Lv1 include: the lack of a well characterized
Rhesus cDNA expression library, the more reliable representation
of low frequency genes, the potential toxicity of over expressing
Lv1 and the potential to isolate non-protein Lv1 products
including RNA.
We will attempt to isolate Rhesus Lv1 by transfecting human
293T cells with a Rhesus genomic cosmid/episomal library DNA.
Cosmid DNA which expresses Lv1 from its native genetic regulatory
elements will selected for by isolating transfected 293T cells
which are relatively resistant to infection with a VSV-G pseudotyped
HIV reporter construct. Cosmids expressing Lv1 would be maintained
in an episomal state by virtue of the presence of the SV40
origin of replication in the cosmid vector, and the expression
of the SV40 large T antigen by 293T cells. Episomal DNA will
be isolated from uninfected 293T cells, amplified in E. coli,
and then tranfected again into 293T cells. A population of
episomes expressing Lv1 will be enriched through repeated
rounds of this selection procedure. Preliminary results using
are presented, which provide evidence for the expression of
an Lv1-like factor in the 293T cells transfected with total
Rhesus genomic DNA. Initial progress with the construction
of a rhesus genomic cosmid/episomal library is described.
The isolation and characterization of Rhesus Lv1 will enhance
our understanding of HIV replication, and provide new targets
for HIV drug design.
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