RNA Content of Retrovirus Particles; Role of RNA in Retrovirus Assembly Immature virion structure is maintained by Gag-RNA as well as Gag-Gag interactions. ?- particles, which assemble efficiently when wild-type Gag is in cells lacking genomic RNA, contain cellular mRNA instead of viral RNA. To clarify mechanisms in specific packaging, we are investigating, for both HIV-1 and Moloney murine leukemia virus (MLV), whether or not these mRNAs are a selected subset of the cellular mRNA. Physical Structure of Genomic RNA Within Retrovirus Particles Retroviral genomic RNAs are dimeric; dimer stabilization accompanies virus maturation. We have localized the strongest linkage in immature MLV RNA to nt 1-754. It requires the "kissing loop," but ?+ RNAs are packaged in pairs even if they cannot form this structure. We also discovered a weaker long-range interaction between regions flanking nt 754. We are currently mapping this interaction, exploring its functional significance, and testing for it in HIV-1 virion RNA. Analysis of Interactions of HIV-1 Proteins with Nucleic Acids in Vitro Both Gag and nucleocapsid (NC) engage in multiple types of functional interactions with nucleic acids. Interactions of NC with oligonucleotides are remarkably complex; it can bind two short oligonucleotides simultaneously. HIV-1 Gag, like NC, is a nucleic acid chaperone; this presumably reflects their ability to reduce cooperativity of base-pair melting in double-stranded DNA. Using fluorescence anisotropy and single-molecule DNA stretching, we are analyzing the binding of Gag to nucleic acids for comparison with NC. We are also investigating whether VLP assembly at high ionic strength favors selectivity in packaging of ?+ RNA in vitro. Search for Inhibitors of Interactions Between HIV-1 Proteins and Nucleic Acids Interactions of both Gag and NC with nucleic acids are crucial in virus replication. We screened a chemical library for inhibitors of these interactions, including compounds that could disrupt VLPs. Several promising chemotypes were identified; these compounds inhibit HIV-1 replication in cell culture at doses 5 ?M and have no effect on MLV replication. We will determine their mechanism of action and will extend our screen to larger chemical libraries.