HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery. We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1. Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III. AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function. These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.

HIV-1 Nef and the unrelated murine leukemia virus glycoGag strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins SERINC3 and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of SERINC3 together with SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivities. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4+ T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

The effects on human immunodeficiency virus type 1 virion morphogenesis and on virus replication of mutations that affect posttranslational processing of the capsid precursor protein are described. A change in the glycine residue at position two from the N terminus abolishes the myristoylation of the precursor proteins and also prevents virus particle release. Mutations in the viral protease gene abolish proteolytic cleavage of the capsid precursor but do not prevent the formation and budding of virion particles of immature appearance. Mutations that alter the sequence of the sites normally used for cleavage of the major capsid protein p24 from the capsid precursor alter virion morphogenesis and prevent virus replication.Electron microscopy of infected cells reveals that particle formation of the human immunodeficiency virus type 1 (HIV-1) begins when an electron-dense structure assembles in patches under the cell membrane (1-3). As the structure assumes a more spherical shape, a portion of the cell membrane pinches off to surround the budding structure. During the course of budding, no clear central core of the virus particle is apparent. After release from the cell, a coneshaped inner core particle is formed (2).What features of the capsid proteins and their precursor polypeptides determine this maturation process? The precursor of the virion capsid proteins is a 55-kDa polyprotein (p55) encoded by the gag region of the viral genome (4). A 160-kDa polyprotein, which is made as a consequence of a -1 frameshift between the gag and pol frames (5), contains precursor forms of the enzymes necessary for virus replication in addition to the capsid proteins. The N terminus ofboth the 55-kDa and 160-kDa proteins is myristoylated (6). Addition of myristic acid to proteins typically occurs cotranslationally (7) and is specified by the sequence ofthe N terminus of the protein (8).Cleavage of both the capsid precursor to yield the mature p17, p24, p9, and p6 capsid proteins as well as the precursor of the replication activities is at least in part specified by the viral protease (9, 10). The viral protease is encoded by the N terminus of the pol reading frame. The protease is reported to be active only as a dimer (11) and has been demonstrated to exhibit a preference for certain natural cleavage sites over others (12).The effects of mutations that prevent the capsid protein myristoylation and the protease activity of HIV-1 on virus assembly and virion morphology have not been reported. The experiments reported here reveal the effects on capsid protein processing, virion morphogenesis, and virus replication of a series of mutations that prevent myristoylation, inactivate the viral protease, or alter the sequence of the cleavage sites. MATERIALS AND METHODSConstruction of Mutants. A 1.3-kilobase (kb) Sac I-Apa I gag fragment from pHXB-SV (13) was subcloned into pBluescriptSK(+) (Stratagene) to generate pSK'gag as a target plasmid for the mutagenesis of the myristoylation site and the N-terminal p24 cleavage site. A 4...