One of the hallmarks of infections with HIV-1 is a long asymptomatic period characterized by low levels of circulating virus. Mechanisms that have been postulated to maintain this clinical latency include immune surveillance and restriction of viral replication it is unknown whether this increase results from the loss of immune modulation, upregulation of virus production within individual cell or both. A key feature of the life cycle of HIV-1 is integration into the host's genome. Once integration takes place, it might be expected that the cell can reorganize and modulate this small gene cluster much as its does the vast number of other gene clusters within its genome. One mechanism which has been postulated to contribute to tissue specific expression/repression of cellular genes is histone mediated assembly of DNA into nucleosomes. Beyond local nucleosome formation, chromatin structural domains have also been demonstrated to influence tissue specific expression of cellular genes. We have shown upregulation of LTR-driven CAT expression as well as p24 antigen production in response to an agent known to disrupt nucleosomes. We have shown this response is not dependent upon de novo protein synthesis; requires at least 12 hours of exposure; is quickly reversible upon its removal; does not map to a specific discrete region; acts synergistically with agents that increase NF-kB expression; mediates a shift in mRNA expression from multiply spliced to full length mRNA; and this effect can be blocked by inhibitors of topoisomerase II. We have demonstrated protection from micrococcal nuclease digestion within the LTR and have cloned and sequenced the integration site of one clone which contains a putative matrix attachment region. These observations are compatible with our hypothesis that there are multiple levels of transcriptional regulation which a cell can bring to bear on either native cellular genes or integrated proviral genes. One such level of regulation is inducible sequence nucleosomes either by directly excluding these sequence specific transcriptional factor or by modifying the torsional stresses within DNA; and further modified by higher order chromatin loops or domains may be limited (or propagated) by torsional stresses on local stretches of DNA under the influence of topoisomerase activity located within the nuclear matrix at these attachment sites. In this application we propose to examine the role of chromatin structure in the modulation of HIV-1 gene expression specifically examining the roles of (i) nucleosome formation, (ii) topoisomerase activity and (iii) nuclear matrix attachment sites.
