Project Summary The HIV-1 RNA genome contains cis-acting RNA sequences elements that control many aspects of viral replication. In addition, there are remarkable global compositional features of the HIV-1 RNA genome that are heretofore unexplained. An essential feature of the HIV-1 RNA genome that enables its encapsidation is its ability to bind to the nucleocapsid (NC) domain in Gag. While a specific interaction between the packaging sequence at the 5' end of the viral genome and Gag is well known to be important for initiating HIV-1 genome packaging it is also well known that the packaging sequence is not absolutely required, nor is it sufficient for highly selective packaging of HIV-1 RNA. In Aim 1 we will determine whether nucleotide composition and sequence motifs throughout the HIV-1 genome constitute a multipartite packaging signal, and whether this feature, coupled with the changing RNA binding specificity of Gag/NC can account for the remarkably specific packaging of HIV-1 genomes into virions. The Matrix (MA) domain of Gag also binds to RNA, as well as to membranes, via a highly basic surface. However, in the case of MA, ~10 host tRNAs are bound with exceptional specificity in the cytoplasm of infected cells. These findings lead to the hypothesis, to be explored in the ensuing funding cycle, that tRNA regulates HIV-1 assembly dynamics or location. Aim 2 is centered on our recent discovery of an RNA binding protein that causes turnover of RNA molecules based on their nucleotide composition. The unusual composition of the HIV-1 genome appears, in part, to represent an evasion strategy to avoid detection by this this protein. We will determine the molecular mechanisms by which this apparent host defense mechanism functions.