Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.     

Relationship between human immunodeficiency virus type 1 Gag multimerization and membrane binding

The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55(Gag), is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between membrane binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the membrane-binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55(Gag) was truncated at the C terminus of matrix (MAstop), between the N- and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using membrane flotation centrifugation, we observe that MA shows significantly reduced membrane binding relative to full-length Gag but that CA146 displays steady-state membrane-binding properties comparable to those of Pr55(Gag). The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state membrane binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient binding of HIV-1 Gag to membrane.     

Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism.

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction.     

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.     

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.     

Myristate exposure in the human immunodeficiency virus type 1 matrix protein is modulated by pH

Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His?Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered?myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a “pH sensor” in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.     

Human immunodeficiency virus type 1 Vpu protein is an oligomeric type I integral membrane protein.

The human immunodeficiency virus type 1 Vpu protein is a 16-kDa phosphoprotein which enhances the efficiency of virion production and induces rapid degradation of CD4, the cellular receptor for human immunodeficiency virus. The topology of membrane-inserted Vpu was investigated by using in vitro-synthesized Vpu cotranslationally inserted into canine microsomal membranes. Proteolytic digestion and immunoprecipitation studies revealed that Vpu was a type I integral membrane protein, with the hydrophilic domain projecting from the cytoplasmic membrane face. In addition, several high-molecular-weight proteins containing Vpu were identified by chemical cross-linking. Such complexes also formed when wild-type Vpu and a Tat-Vpu fusion protein were coexpressed. Subsequent analysis by one- and two-dimensional electrophoresis revealed that these high-molecular-weight complexes consisted of homo-oligomers of Vpu. These findings indicate that Vpu is a type I integral membrane protein capable of multimerization.     

Characterization of human immunodeficiency virus type 1 p17 matrix protein motifs associated with mother-to-child transmission.

In order to determine if viral selection occurs during mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1), we used a direct solid-phase sequencing method to sequence the p17 matrix protein-encoding regions of viral isolates from 12 HIV-1-infected mother-and-child pairs, 4 infected infants, 4 transmitting mothers, and 22 nontransmitting mothers and compared the sequences. The blood samples were collected during the delivery period for the mothers and during the first month of life for most of the children. The p17 nucleic sequences were distributed among several clades corresponding to the HIV-1 A, B, and G subtypes. At the amino acid level, no significant differences within the known p17 functional regions were observed among the subtypes. Statistical analyses could be performed with the B subtype. Within the major p17 antibody binding site, a constant KIEEEQN motif (amino acids 103 to 109) was found in all mother-and-child isolates from the B subtype. On the other hand, 9 of 17 nontransmitting mother isolates were variable in this 103 to 109 region. Thus, this motif was significantly associated with the transmitting status (chi square, P = 0.0034). A valine residue at position 104 was significantly associated with the nontransmitting phenotype (chi square, P = 0.014), suggesting that it has a protective role during vertical transmission. The C-terminal end of p17 was globally conserved among nontransmitting mother isolates (chi square, P = 0.0037). These results might improve the understanding of the pathogenesis of HIV-1 vertical transmission and might allow the screening of seropositive mothers by a rapid molecular or peptide test.     

Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein.

The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) forms an inner coat directly underneath the lipid envelope of the virion. The outer surface of the lipid envelope surrounding the capsid is coated by the viral Env glycoproteins. We report here that the HIV-1 capsid-Env glycoprotein association is very sensitive to minor alterations in the MA protein. The results indicate that most of the MA domain of the Gag precursor, except for its carboxy terminus, is essential for this association. Viral particles produced by proviruses with small missense or deletion mutations in the region coding for the amino-terminal 100 amino acids of the MA protein lacked both the surface glycoprotein gp120 and the transmembrane glycoprotein gp41, indicating a defect at the level of Env glycoprotein incorporation. Alterations at the carboxy terminus of the MA domain had no significant effect on the levels of particle-associated Env glycoprotein or on virus replication. The presence of HIV-1 MA protein sequences was sufficient for the stable association of HIV-1 Env glycoprotein with hybrid particles that contain the capsid (CA) and nucleocapsid (NC) proteins of visna virus. The association of HIV-1 Env glycoprotein with the hybrid particles was dependent upon the presence of the HIV-1 MA protein domain, as HIV-1 Env glycoprotein was not efficiently recruited into virus particles when coexpressed with authentic visna virus Gag proteins.     

Transcellular transactivation by the human immunodeficiency virus type 1 tat protein.

The human immunodeficiency virus type 1 (HIV-1) transactivator (tat) protein produced in one cell activated HIV-1 promoter-directed gene expression in a second cell, provided the cells were in direct contact with one another. This observation suggests that the tat protein produced in HIV-1-infected cells has a physiological effect on neighboring cells.     

Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55(Gag), to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55(Gag), a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.     

Functional domains of the capsid protein of human immunodeficiency virus type 1.

A series of deletions was introduced into the CA domain of the human immunodeficiency virus type 1 Gag polyprotein to examine its role in virus particle and core formation. The mutations resulted in two phenotypes, indicating the existence of two functionally distinct regions within the CA domain. Deletions within a conserved stretch of 20 amino acids referred to as the major homology region (MHR) and deletions C terminal to this region blocked virus replication and significantly reduced the ability to form viral particles. Deletions N terminal to the MHR also prevented virus replication, but the mutants retained the ability to assemble and release viral particles with the same efficiency as the wild-type virus. The mutant particles contained circular rather than cone-shaped cores, and while they were of a density similar to that of wild-type particles, they were more heterogeneous in size. These results indicate that CA domain sequences N terminal to the MHR are essential for the morphogenesis of the mature cone-shaped core.     

Subcellular localization of the Vif protein of human immunodeficiency virus type 1.

The Vif (viral infectivity factor) protein of human immunodeficiency virus type 1 (HIV-1) has been shown to dramatically enhance the infectivity of HIV-1 virus particles during virus production. The subcellular localization of Vif was examined to elucidate cellular pathways which may be important for Vif function. Indirect immunofluorescence staining of Vif demonstrated a diffuse cytoplasmic distribution and showed that most Vif was not associated with the Golgi complex, a proposed site of localization (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). Subcellular fractionation of transfected COS cells and HIV-1-infected Jurkat and CEM cells demonstrated that Vif is a cytoplasmic protein which exists in both a soluble cytosolic form and membrane-associated form. The membrane-associated form of Vif is a peripheral membrane protein which is tightly associated with the cytoplasmic side of cellular membranes. The C terminus of Vif was required for the stable association of Vif with membranes. The C terminus was also essential for Vif function, suggesting that the association of Vif with membranes is likely to be important for its biological activity. The highly conserved regions at residues 103 to 115 and 142 to 150 were important for Vif function but did not affect membrane association, indicating that these regions are likely to be important for other, as-yet-unknown functions.     

In vitro assembly of human immunodeficiency virus type 1 Gag protein.

Retroviral Gag protein is sufficient to produce Gag virus-like particles when expressed in higher eukaryotic cells. Here we describe the in vitro assembly reaction of human immunodeficiency virus Gag protein, which consists of two sequential steps showing the optimal conditions for each reaction. Following expression and purification, Gag protein lacking only the C-terminal p6 domain was present as a monomer (50 kDa) by velocity sedimentation analysis. Initial assembly of the Gag protein to 60 S intermediates occurred by dialysis at 4 degrees C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37 degrees C and was facilitated by the addition of Mg(2+). Prolonged incubation under these conditions produced complete assembly (600 S), equivalent to Gag virus-like particles obtained from Gag-expressing cells. Neither form disassembled by treatment with nonionic detergent, suggesting that correct assembly might occur in vitro. Electron microscopic observation confirmed that the 600 S assembly products were spherical particles similar to authentic immature human immunodeficiency virus particles. The latter assembly stage but not the former was accelerated by the addition of RNA although not inhibited by RNaseA treatment. These results suggest that Gag protein alone assembles in vitro, but that additional RNA facilitates the assembly reaction.     

Mutational analysis of the human immunodeficiency virus type 1 Vif protein

Lentivirus Vif proteins are potent regulators of virus infectivity. However, relatively little is known about the functional domains, peptide motifs, or residues of any Vif protein. In this report, we present the first extensive mutagenesis analysis of the 192-amino-acid human immunodeficiency virus type 1 (HIV-1) Vif protein. A large number of scanning missense (mostly alanine substitution) and deletion mutations were introduced into the HIV-1HXB3 vif gene, and the resulting proteins were evaluated for the induction of virus infectivity as well as subcellular localization. The results show that amino acids dispersed throughout Vif's linear sequence are important for function. However, because many of the inactive proteins also appear to be mislocalized, we suggest that many of them may actually be misfolded rather lacking an intracellular targeting signal. Interestingly, disruptions within an internal region spanning residues 114 to 146 give rise to mutant proteins that either retain function or are inactive but are not substantially mislocalized. We therefore speculate that this region, which harbors two essential cysteine residues and one essential serine residue, may contain aspects of a putative Vif effector domain.