Sequence specific RNA binding is thematic of several regulatory mechanisms of HIV gene expression. HIV RNA capsidation and particle maturation are driven by similar biochemical events. Self-assembly of p55 Gag protein is sufficient to form the skeletal framework of the immature particle. Coexpression of the Gag-Pol fusion protein facilitated the maturation and processing as manifested by the condensation of the core. This morphologic change was accompanied by substantial Gag processing in the Gag-Pol particles as evidenced by the presence of abundant p24 (CA) protein and encapsidation of viral RNA. The cooperative interactions regulating capsidation and particle assembly are attractive for targeted drug development. The mechanism of RNA encapsidation during retrovirus particle formation is still unclear. In particular, the nature and the identity of the RNA sequences and the gag subunits involved in HIV capsidation remain to be solved. We have now developed a simple in vivo model for HIV-1 RNA packaging. A subgenomic HIV RNA encompassing R, U5 and U3 elements surrounding the minimal packaging sequence was expressed from the T7 promoter using vaccinia T7 polymerase vector. Co-infection with GAG or GAG-POL recombinant vaccinia viruses lead to HIV particles that selectively packaged the mini HIV RNA. We have expressed cleavage defective mutants of GAG using the vaccinia vector. Mutations that eliminate the myristoylation or the nucleic acid binding CYS containig motif (NBCys) of the p9 subunit of GAG protein have been analyzed in the context of GAG-POL expression to examine whether they interfere with RNA packaging. This system is being used to define the molecular parameters of RNA capsidation. It is our eventual goal to develop molecular strategies to interfere with RNA packaging and particle maturation. GRAMT=Z01AI00527 Heterogeneity among AIDS retroviral genomes is a distinct feature of lentiviruses. Because of this diversity, molecular clones provide the necessary molecular reagents for identifying and studying the functions of the viral genome. These clones can also be used to characterize viral determinants of cell tropism, cytopathicity, and virulence in cultured cells. Chimeric constructs between these molecular clones can be used to examine the function of individual genes as well as entry and replication. Our objective was to obtain information pertaining to the structure and diversity of HIV. Most HIV isolates cloned are T cell tropic. We have a complete molecular clone from a macrophagetropic viral isolate. Although T cells are the major target for HIV replication in peripheral blood, macrophages represent the predominant HIV-infected cell type in most tissues. Macrophages are probably the primary reservoir of HIV and sustain a persistent infection in individuals for many years. Our macrophagetropic clone (PAD-2), upon transfection into Hela cells followed by cocultivation on PBLs or macrophages, produced viral particles as measured by reverse transcriptase (RT) activity. This clone was unable to replicate in continuous T cell lines. Growth studies, as well as physical and biochemical analyses with this and other isolates, are in progress.
