Following infection of target cells, retroviral nucleic acids are synthesized and transported to the host cell nucleus in the context of a high molecular weight (approximately 160S) nucleoprotein """"""""preintegration"""""""" complex. For onco-retroviruses--such as MLV-- the preintegration complex is unable to translocate efficiently across the nuclear envelope until nuclear envelope breakdown during host cell mitosis. In contrast, lentiviruses--such as HIV-1--infect efficiently non-dividing cells. The Principal Investigator and his associates have demonstrated that this property of HIV is governed by nucleophilic virion components (including gag MA and vpr) which facilitate the nuclear localization of viral preintegration complexes in the absence of host cell mitosis. The nuclear import function of HIV-1 gag MA in the infection of non-dividing cells creates a paradox. During virus assembly, gag MA is cotranslationally myristylated: a modification necessary for efficient membrane targeting of gag polyproteins in the plasma membrane. During virus infection, however, gag MA is rapidly targeted to the host cell nucleus within the context of viral preintegration complexes. The Investigator's recent studies demonstrate that phosphorylation of gag MA is required to allow membrane dissociation of gag MA at the site of virus entry, thus allowing its nuclear import function to operate. Thus, the Investigator proposes that phosphorylation provides a molecular switch that regulates the nuclear import and membrane targeting properties of gag MA and, this, is a central event governing HIV-1 infectivity. In the studies outlined in this AIDS-FIRCA application, the Investigator and his collaborator will examine specifically how phosphorylation of gag MA alters subcellular distribution of the viral preintegration complex in acutely infected cells. These studies , which will be undertaken entirely in the collaborator's laboratory, will benefit from the productivity of the collaboration to date and the expertise of individuals in the collaborator's laboratory in techniques related to fractionation of viral nucleoprotein complexes and their biochemical analysis.
Their aims are to examine: (1) the subcellular distribution of viral preintegration complexes in cells following infection with hypophosphorylated HIV-1 virions; and (2) the subcellular distribution of viral preintegration complexes in cells infected with defined phosphate acceptor site mutants of HIV-1. The proposed studies will complement the Investigator's parent grant by providing evidence for the role of phosphorylation in the infectivity of HIV-1.
