Human cells resist viral infections by a variety of mechanisms.Viruses must overcome host cell restrictions to successfully reproduce their genetic material. Here, we identify a host restriction to viral replication that acts at the stage of particle assembly. Viral protein U (Vpu) is an HIV-1 accessory protein that enhances particle assembly and release in most human cells, but not in simian cells. By using human-simian cell heterokaryons, we show that the inhibition of assembly in human cells is dominant. Vpu overcomes the block to assembly in human cells and in human-simian heterokaryons. The HIV-1 vpu gene may have evolved to counteract an assembly restriction that is present in human cells.

Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55Gag molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55 Gag . The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.

The HIV‐1 accessory gene product Vpu is required for efficient viral particle release from infected human cells. The mechanism by which Vpu enhances particle assembly or release is not yet defined. Here, we identify an intracellular site that is critical for Vpu‐mediated enhancement of particle release. Vpu was found to co‐localize with markers for the pericentriolar recycling endosome. Expression of dominant negative mutants of Rab11a and myosin Vb that disrupt protein sorting through the recycling endosome abrogated the ability of Vpu to augment particle release. Remarkably, the effects of blocking recycling endosome function on HIV particle release were demonstrable only in human cell lines known to be responsive to Vpu, while no effect on particle release was seen in African green monkey cells. Inhibition of recycling endosome function in human cells also blocked the ability of HIV‐2 envelope to enhance particle release. These studies indicate that Vpu and HIV‐2 envelope glycoprotein enhance particle release via a common mechanism that requires the activity of the pericentriolar recycling endosome.