The goal of this proposal is to identify and characterize the elements that allow HIV-l to achieve selective and efficient encapsidation of its genomic RNA during particle morphogenesis. A basic understanding of these critical molecular interactions could lead to the definition of new targets for drug development and is expected to help in the design of HIV-1 based vectors for the transfer of antiviral agents to the target cells of HIV-1. Only a small portion of the HIV-l genome has thus far been analyzed for the presence of packaging recognition sequences. Our preliminary data and indirect evidence from others suggest that important cis-acting elements for encapsidation are located upstream of the major splice donor site and downstream of the gag initiation site. Our data also support the possible relevance of a recently proposed secondary structure that involves sequences on both sides of the splice donor. The functional importance of this putative structure for encapsidation will be investigated. We have demonstrated that the gene products required for encapsidation can be provided in trans, which will permit a mutational analysis of the gag-region for encapsidation signals. A highly sensitive RNase protection assay was developed for the detection of viral RNA in mutant particles. The HIV-1 genome consists of a dimer of identical RNA molecules. Evidence exists that encapsidation and dimerization may be linked events. The possibility that encapsidation signals also serve as signals for dimerization will be analyzed. To determine which of the domains of the HIV-1 capsid precursor is involved in specific RNA recognition during encapsidation, the properties of chimeric capsid precursors will be analyzed. We have demonstrated that chimeric precursors can efficiently form particles by replacing the matrix domain of the visna capsid precursor with that of HIV-1. We have also shown that visna particles do not efficiently package HIV-1 RNA. Therefore, it is anticipated that the analysis of the packaging specificity of chimeric precursors will reveal the role of individual domains in selective packaging.
