Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immunodeficiency syndrome (AIDS) and is estimated to infect roughly one million Americans. Currently available therapies for HIV-infected individuals are inadequate. We propose to develop a novel gene delivery system, which would allow for """"""""intracellular immunization"""""""" against HIV in vivo. Human herpesvirus 6 (HHV-6) is a ubiquitous virus with minimal known pathogenicity, and a host cell range very similar to that of HIV-1 (it infects CD4+ T-cells and monocyte/macrophages). This proposal sets forth experiments that are designed to characterize cis-acting sequences which regulate DNA replication of HHV-6 with a view towards developing HHV-6 based vectors. By analogy to other herpesviruses, it is expected that two DNA elements will be required for HHV-6 replication. These comprise an origin of replication and a packaging site. Using a transient replication-complementation assay it has been shown that plasmid constructs containing cloned fragments of HHV-6 DNA were replicated in human T-cells when trans-acting factors were provided by HHV-6 infection. Thus, a lytic-phase origin of HHV-6 DNA replication has been identified. A viral packaging site will identified by a similar approach and both of these elements will be molecularly characterized via-site-directed mutagenesis. These DNA elements will then be combined into a single plasmid, or """"""""amplicon"""""""", together with a beta-galactosidase indicator gene. The ability of this construct to mediate gene transfer into various cells will be assessed, using wild-type helper virus. Studies will also be carried out to assess the feasibility of constructing HHV-6 based human artificial chromosomes. The genomic status of HHV-6 DNA during viral latency will be determined, and the functional significance of telomere-like structures within the HHV-6 genome will be studied. 2D-gels and stable replication assays will then be used to determine whether a distinct viral origin, analogous to EBV oriP, is used during latent-phase HHV-6 DNA replication. These studies are expected to contribute to the development of a novel gene delivery system for intracellular immunization against HIV-1.
