Alternative splicing of RNA is a mechanism that is used to regulate gene expression during cell growth and differentiation. The role of this mechanism has been examined in regulating the replicative capacity of HIV. Many of the splice donor and acceptor motifs in the infectious clone of HIV, pNL4-3, have been mutated, yielding viruses that fail to produce the full complement of spliced mRNAs. Analyses of the biological and biochemical properties of these viruses indicate that they exhibit patterns of protein expression and altered growth kinetics despite the apparent redundancy of splicing signals. A semi-quantitative PCR assay has been developed that determines the relative proportions of alternatively spliced HIV mRNA species during infection. This assay is being used in conjunction with mutagenesis studies to examine HIV mRNA expression in different cell types and correlate the expression of alternatively spliced mRNAs with the biological and biochemical properties of HIV generated in these cells. A sensitive system to monitor the cell-to-cell transmission of HIV-1 has been developed which utilizes chronically infected H9 donor cells. Studies with this system have shown that virus induced cell-cell fusion occurs within 15 to 30 minutes of co-culture as monitored by electron microscopy, sCD4 competition and dye-diffusion analysis. This system has also been used to evaluate HIV-1 infection of activated and resting human PBLs. Activated unfractionated PBLs synthesize viral DNA 4 to 6 hrs. following incubation with infected H9 cells and produce progeny virions by 22 hrs. post infection. Viral DNA is also produced in resting unstimulated PBLs (to approximately 40% the level observed in activated cells) but no detectable progeny virions are produced. Linear as well as circular forms of viral DNA are synthesized in resting PBLs indicating that the nuclear transport of reverse transcripts is not inhibited. Studies are underway to ascertain whether the integration step of the virus life cycle is blocked in unstimulated PBLs.
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