Characterization of human immunodeficiency virus type 1 Vif particle incorporation.

The human immunodeficiency virus type 1 (HIV-1) Vif protein is necessary at the time of viral particle formation yet functionally manifests its effect after virions enter target cells. This suggests that Vif either acts on another viral protein or is itself incorporated into particles. In this study, we have examined the latter possibility. We confirm our previous observation that Vif is incorporated into human immunodeficiency virus type 1 virions at a ratio of approximately 1 molecule of Vif for every 75 to 220 molecules of p24, or 7 to 20 molecules per virion. Furthermore, we demonstrate that the relative concentration of Vif is much lower in particles than in infected cells, whereas the opposite is observed for the main virus components. The viral envelope, Nef, Vpr, Vpu, protease, reverse transcriptase, integrase, nucleocapsid, and p6gag proteins as well as the viral genomic RNA are dispensable for Vif packaging. Furthermore, mutating several highly conserved residues (H-108, C-114, C-133, L-145, and Q-146) or deleting the C-terminal 18 amino acids of Vif, either of which severely impairs Vif function, does not abolish its incorporation into virions. Finally, Vif can be packaged into murine leukemia virus particles. On the basis of these data, we conclude that the specificity of Vif incorporation into virions remains an open question

The proteolytic cleavage of human immunodeficiency virus type 1 Nef does not correlate with its ability to stimulate virion infectivity

The Nef protein of human immunodeficiency virus type 1 (HIV-1) promotes virion infectivity through mechanisms that are yet ill defined. Some Nef is incorporated into particles, where it is cleaved by the viral protease between amino acids 57 and 58. The functional significance of this event, which liberates the C-terminal core domain of the protein from its membrane-associated N terminus, is unknown. To address this question, we examined the modalities of Nef virion association and processing. We found that although significant levels of Nef were detected in HIV-1 virions partly in a cleaved form, cell-specific variations existed in the efficiency of Nef proteolytic processing. The virion association of Nef was strongly enhanced by myristoylation but did not require other HIV-1-specific proteins, since Nef was efficiently incorporated into and cleaved inside murine leukemia virus particles. Substituting alanine for tryptophan57 decreased the efficiency of Nef processing, while mutating leucine58 had little effect. In contrast, replacing both of these residues simultaneously almost completely prevented this process. However, when the resulting mutants were compared with a wild-type control in viral infectivity assays, no correlation was found between the levels of cleavage and the ability to stimulate virion infectivity. Furthermore, simian immunodeficiency virus Nef, which lacks the sequence recognized by the protease and as a consequence is not cleaved despite its incorporation into virions, could stimulate the infectivity of a nef-defective HIV-1 variant as efficiently as HIV-1 Nef. On these bases, we conclude that the proteolytic processing of Nef is not required for the ability of this protein to enhance virion infectivity

Human immunodeficiency virus matrix tyrosine phosphorylation: characterization of the kinase and its substrate requirements

During virus assembly, a subset of human immunodeficiency virus (HIV) matrix (MA) molecules is phosphorylated on C-terminal tyrosine. This modification facilitates infection of nondividing cells by allowing for the recruitment of the karyophilic MA into the viral core and preintegration complex. MA tyrosine phosphorylation is accomplished by a cellular protein kinase which is incorporated into virions. In this study, we have investigated the nature of this enzyme as well as the determinants of MA necessary for its phosphorylation. Employing an in vitro kinase assay, we found that the MA tyrosine kinase activity is present in various cultured cell lines including CEM and SupT1 T-lymphoid cells, Namalwa B cells, 293 and CV-1 kidney fibroblasts, and P4 HeLa cells. In addition, it could be detected in platelets, macrophages, and activated peripheral blood lymphocytes (PBLs) but not in erythrocytes and resting PBLs isolated from human blood. Subcellular localization of the kinase activity by cell fractionation demonstrated that it is enriched in cellular membranes. In HIV type 2 (HIV-2) particles, the MA tyrosine kinase is associated with the inner leaflet of the viral membrane, while the tyrosine-phosphorylated MA is localized to the core. Individual mutations of each of the last eight residues immediately upstream of the C-terminal tyrosine (Y132) of HIV-1 MA did not prevent Y132 phosphorylation, suggesting that the kinase does not require a highly specific sequence adjacent to the C-terminal tyrosine. Confirming this, we found that the MA of murine leukemia virus, the sequence of which is only moderately homologous to that of HIV-1 and HIV-2 MA, is also C-terminally tyrosine phosphorylated

The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase