Analysis of human immunodeficiency virus type 1 Gag ubiquitination

Ubiquitin is important for the release of human immunodeficiency virus type 1 (HIV-1) and several other retroviruses, but the functional significance of Gag ubiquitination is unknown. To address this problem, we decided to analyze Gag ubiquitination in detail. A low percentage of the HIV-1 p6 protein has previously been shown to be ubiquitinated, and published mutagenesis data suggested that Gag ubiquitination is largely lost upon mutation of the two lysine residues in p6. In this study, we show that Gag proteins lacking the p6 domain or the two lysine residues within p6 are ubiquitinated at levels comparable to those of the wild-type Gag protein. We detected monoubiquitinated forms of the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins in mature virus preparations. Protease digestion of Gag polyproteins extracted from immature virions indicated that ubiquitinated MA, CA, and possibly NC are as abundant as ubiquitinated p6. The HIV-1 late-domain motifs PTAP and LRSLF were not required for Gag ubiquitination, and mutation of the PTAP motif even resulted in an increase in the amount of Gag-Ub conjugates detected. Finally, at steady state, ubiquitinated Gag proteins were not enriched in either membrane-associated or virus-derived Gag fractions. In summary, these results indicate that HIV-1 Gag can be monoubiquitinated in all domains and that ubiquitination of lysine residues outside p6 may thus contribute to viral release and/or infectivity.     

Analysis of human immunodeficiency virus type 1 Gag dimerization-induced assembly

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.     

Ubiquitination of human immunodeficiency virus type 1 Gag is highly dependent on Gag membrane association

Ubiquitin is important for the release of human immunodeficiency virus 1 (HIV-1) and several other retroviruses. All major domains of the HIV-1 Gag protein are monoubiquitinated, but the modifying machinery and the function of HIV-1 Gag ubiquitination remain unclear. Here, we show that the induction of a late budding arrest by mutation of the HIV-1 PTAP motif or by specific inhibition of selected ESCRT components leads to an increase of Gag-ubiquitin conjugates in cells, which coincides with an accumulation of detergent-insoluble, multimerized Gag at the plasma membrane. Membrane flotation experiments revealed that ubiquitinated Gag is highly enriched in membrane-bound fractions. Based on these findings, we propose that a blocking of virus release results in increased Gag ubiquitination as a consequence of its prolonged membrane association. Consistent with this, ubiquitination of a membrane-binding-defective (G2A)Gag mutant was dramatically reduced and the ubiquitination levels of truncated Gag proteins correlated with their abilities to bind to membranes. We therefore propose that membrane association and multimerization of HIV-1 Gag proteins, rather than a specific motif within Gag, trigger recognition by the cellular ubiquitination machinery.     

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.     

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.     

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.     

Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6(Gag) or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.     

Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells

Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.     

Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.     

Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes

Assembly of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein on budding virus particles is important for efficient infection of target cells. In infected cells, lipid rafts have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with detergent-resistant membranes (DRMs) that are believed to represent lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer DRM association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific Gag-envelope interactions. Thus, specific mutations in both the matrix domain of the Gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRMs is directly influenced by its interaction with Gag. Thus, in the absence of Gag, envelope fails to associate with DRMs. A mutation in the p17 matrix (L30E) domain in Gag (Gag L30E) that abrogates envelope incorporation onto virions also eliminated envelope association with DRMs in 293T cells and in the T-cell line, MOLT 4. These observations are consistent with a requirement for an Env-Gag interaction for raft association and subsequent assembly onto virions. In addition to this observation, we found that mutations in the gp41 cytoplasmic domain that abrogated envelope incorporation onto virions and impaired infectivity of cell-free virus also eliminated envelope association with DRMs. On the basis of these observations, we propose that Gag-envelope interaction is essential for efficient envelope association with DRMs, which in turn is essential for envelope budding and assembly onto virus particles.     

Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions.

The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.     

Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages

Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM.     

Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA

Retroviral Gag polyproteins drive virion assembly by polymerizing to form a spherical shell that lines the inner membrane of nascent virions. Deletion of the nucleocapsid (NC) domain of the Gag polyprotein disrupts assembly, presumably because NC is required for polymerization. Human immunodeficiency virus type 1 NC possesses two zinc finger motifs that are required for specific recognition and packaging of viral genomic RNA. Though essential, zinc fingers and genomic RNA are not required for virion assembly. NC promiscuously associates with cellular RNAs, many of which are incorporated into virions. It has been hypothesized that Gag polymerization and virion assembly are promoted by nonspecific interaction of NC with RNA. Consistent with this model, we found an inverse relationship between the number of NC basic residues replaced with alanine and NC's nonspecific RNA-binding activity, Gag's ability to polymerize in vitro and in vivo, and Gag's capacity to assemble virions. In contrast, mutation of NC's zinc fingers had only minor effects on these properties.     

A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41

The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55(Gag). Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity.     

A heterologous, high-affinity RNA ligand for human immunodeficiency virus Gag protein has RNA packaging activity

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (psi) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 psi locus to support packaging and viral infectivity in cis. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different psi element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.