Insights Into The Mechanism Of Retroviral Genome Packaging And Assembly

Abstract

In HIV-1 infected cells, newly synthesized retroviral Gag polyproteins are directed to specific cellular membranes where they assemble and bud to form immature virions. Membrane binding is mediated by Gag's matrix (MA) domain, a 132-residue polypeptide containing an N-terminal myristyl group that can adopt sequestered and exposed conformations. Membane specificity was recently shown to be regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), a cellular factor abundant in the inner leaflet of the plasma membrane (PM). We now show that phosphoinositides, including soluble analogs of PI(4,5)P2 with truncated lipids, bind HIV-1 MA and trigger myristate exposure. The phosphoinositol moiety and one of the fatty acid tails binds to a cleft on the surface of the protein. The other fatty acid chain of PI(4,5)P2 and the exposed myristyl group of MA bracket a conserved basic surface patch implicated in membrane binding. Our findings indicate that PI(4,5)P2 acts as both a trigger of the myristyl switch and as a membrane anchor, and suggest a structure-based mechanism for the specific targeting HIV-1 Gag to PI(4,5)P2-enriched membranes. Retroviral genomes contain elements within their 5′-untranslated regions (UTRs) that regulate multiple essential functions, including splicing, nuclear export, translational activation, genome packaging, and reverse transcription, among others. A number of studies suggest that these processes may be differentially regulated by RNA conformational changes. To gain insights into the structural basis for these processes, we have initiated NMR studies of intact retroviral packaging elements, including the native, dimeric 200 nucleotide core encapsidation signal (ΨCES) of the Moloney murine leukaemia virus (MLV) and the intact, dimeric 748 nucleotide 5′-UTR of the human immunodeficiency virus Type-1 (HIV-1). Progress toward the implementation of these data as restraints for structure refinement of the dimeric MLV ΨCES will be presented.