Human immunodeficiency virus type 1 protease triggers a myristoyl switch that modulates membrane binding of Pr55(gag) and p17MA.

The assembly of lentiviruses and type C retroviruses occurs at the plasma membrane and is directed by the viral gag gene product (34). In human immunodeficiency virus type 1 (HIV-1), Pr55gag contains an N-terminal myristoyl group and a cluster of positively charged residues that act synergistically to promote plasma membrane targeting (37, 38). A combination of a myristate and a basic residue cluster also functions in the Src tyrosine kinase and in the MARCKS protein as a plasma membrane binding motif (5, 29). In addition to plasma membrane binding, the functional properties of Pr55gag responsible for the production of an infectious virion include recruitment of the HIV-1 genome and specific viral proteins into budding virions and processing by HIV-1 protease to generate the structural proteins of the mature virus (8, 17, 18, 21, 22, 36). The process of viral maturation occurs during or immediately following retroviral assembly (19) and involves proteolysis of the Gag protein by the virus-encoded protease. In HIV-1, processing of Pr55gag by protease generates the following viral structural proteins (from N to C terminus): matrix (p17MA), capsid (p24CA), nucleocapsid (p7NC), and p6 (14). Two small peptides, p2, located between p24CA and p7NC, and p1, located between p7NC and p6, have also been described (1). Additionally, maturation involves processing of Pr160gag-pol, a polypeptide synthesized as a result of ribosomal frameshifting and produced at about 5% the level of Pr55gag. Maturation of Gag-Pol generates the retroviral enzymes protease, reverse transcriptase, and integrase. The localization of each of the products of Pr55gag within the virion has been determined (13). The matrix protein p17MA forms a shell immediately underneath the viral membrane envelope. The capsid protein polymerizes to give rise to the conical structure inside mature viral particles. The nucleocapsid is found associated with the viral genome within the mature viral core, which carries two copies of RNA per viral particle. The function of the p2 spacer peptide is not well understood, but it appears to be essential for the correct morphology of the viral core (1, 26). p6 appears to be essential for the release of the viral particle from the infected cell (21). The matrix protein p17MA also plays a role during the early stages of the HIV-1 viral life cycle. After viral infection and fusion with the cell plasma membrane, a portion of p17MA dissociates from the membrane. A subpopulation is phosphorylated and transported into the nucleus as a component of the preintegration complex (32). Thus, the matrix region of Gag displays different subcellular localizations, depending on the stage of viral infection. In a previous report from our laboratory, the molecular basis for the differential membrane binding of Pr55gag and p17MA was examined (39). The intrinsic affinity of p17MA for membranes was shown to be three- to fivefold lower than that of Pr55gag, despite the fact that both proteins contain the same combination of N-terminal myristate and basic residues. Sequential removal of alpha-helical regions of p17MA restored plasma membrane binding (39). It was proposed that the myristate moiety was exposed within the context of Pr55gag but became sequestered within p17MA. This proposal is consistent with a myristoyl switch mechanism in which a conformational change regulates exposure of the myristate moiety and the membrane binding affinity of p17MA during the viral life cycle. A similar conclusion was reached by other investigators (30). The myristoyl group within p17MA can be artificially reexposed by internal or C-terminal deletions within p17MA (30, 39). However, we propose that the physiological “trigger” for the myristoyl switch is cleavage of Pr55gag by HIV-1 protease. Here, we test this hypothesis by studying the membrane binding properties of newly generated p17MA derived directly from Pr55gag by proteolysis with HIV-1 protease. We show that p17MA generated in situ from membrane-bound Pr55gag initially is membrane bound but is slowly released from the membrane into the soluble fraction. Additional experiments with Gag incorporated into sealed right-side-out membrane vesicles provide insight into the nature of the membrane association of p17MA inside the viral particle. These studies effectively reproduce critical steps of viral maturation in the test tube and serve as a model for understanding the regulation of differential membrane binding of HIV-1 Gag.