The C-Terminal Half of the Human Immunodeficiency Virus Type 1 Gag Precursor Is Sufficient for Efficient Particle Assembly

Human immunodeficiency virus type 1 particle assembly is directed by the Gag polyprotein Pr55^gag, the precursor for the matrix (MA), capsid (CA), and nucleocapsid proteins of the mature virion. We now show that CA sequences N terminal to the major homology region (MHR), which form a distinct domain, are dispensable for particle formation. However, slightly larger deletions which extend into the MHR severely impair particle production. Remarkably, a deletion which removed essentially all MA and CA sequences between the N-terminal myristyl anchor and the MHR reduced the yield of extracellular particles only moderately. Particle formation even exceeded wild-type levels when additional MA sequences, either from the N or the C terminus of the domain, were retained. We conclude that no distinct region between the myristyl anchor and the MHR is required for efficient particle assembly or release.     

Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55gag and p17MA

The human immunodeficiency virus type 1 (HIV-1) Pr55^gag gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55^gag is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55^gag maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55^gag in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55^gag was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55^gag was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55^gag and p17MA in virions and membranes.     

Analysis of the Assembly Function of the Human Immunodeficiency Virus Type 1 Gag Protein Nucleocapsid Domain

Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55^gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55^gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.Human immunodeficiency virus type 1 (HIV-1) encodes three major genes, gag, pol, and env, which are commonly found in all mammalian retroviruses. It also encodes accessory genes whose protein products are important for regulation of its life cycle (6, 30, 35). However, of all the genes encoded by HIV-1, only the protein product of the gag gene has been found to be necessary and sufficient for the assembly of virus-like particles (11, 13, 17, 22, 32, 33). The HIV-1 Gag protein initially is expressed as a 55-kDa polyprotein precursor (Pr55^gag), but during or shortly after particle release, Pr55^gag ordinarily is cleaved by the viral protease (PR). The products of the protease action are the four major viral proteins matrix (MA), capsid (CA), nucleocapsid (NC), and p6, and the two spacer polypeptides p2 and p1, which represent sequences between CA and NC and between NC and p6, respectively (15, 19, 23, 30).The HIV-1 nucleocapsid proteins have two Cys-X₂-Cys-X₄-His-X₄-Cys (Cys-His) motifs, reminiscent of the zinc finger motifs found in many DNA binding proteins, and NC has been shown to facilitate the specific encapsidation of HIV-1 genomic RNAs. In addition to its encapsidation function, NC influences virus particle assembly (7, 10, 17, 21, 40). In particular, Gag proteins lacking the NC domain fail to assemble virus particles efficiently. Nevertheless, some chimeric Gag proteins which carry foreign sequences in place of NC have been shown to assemble and release virus particles at wild-type (wt) levels (2, 37, 40). Thus, it appears that in some circumstances, the role that NC plays in virus particle assembly can be replaced. To date, it is not clear how NC affects particle assembly, although several possibilities might be envisioned. One possibility is that deletion of NC unmasks inhibitory sequences in p2 or the C terminus of CA. Alternatively, NC may simply provide a stable monomeric folded structure which locks CA or other Gag domains into an assembly-competent conformation. Another possibility is that NC facilitates assembly by forming essential protein-protein contacts between neighbor Pr^gag molecules, as suggested in cross-linking studies (21). Finally, the assembly role of NC may stem from its RNA binding capabilities, a hypothesis supported by studies of Campbell and Vogt (5), which have shown that RNA facilitates the in vitro assembly of retroviral Gag proteins into higher-order structures.To distinguish among possible mechanisms by which NC facilitates HIV-1 assembly, we replaced NC with polypeptides having known structural characteristics and examined particle assembly directed by these chimeric proteins. Using this approach, we have found that NC does not play a passive role in HIV-1 assembly as either a mask to assembly inhibitor domains or a nonspecific, stably folded structure. Rather, sequences known to form strong interprotein contacts were observed to enhance assembly, suggesting a similar role for the NC domain itself. With several assembly-competent chimeric proteins, we detected no particle-associated RNAs. These results suggest that while RNA may be essential to virus assembly in the context of the wt Pr55^gag protein, it is dispensable for formation of virus-like particles from chimeric proteins.     

In Vitro Assembly Properties of Human Immunodeficiency Virus Type 1 Gag Protein Lacking the p6 Domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Δp6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Δp6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Δp6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Δp6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Δp6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P₂. We have recently shown that PI(4,5)P₂ binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P₂. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P₂–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P₂. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.     

Particle Size Determinants in the Human Immunodeficiency Virus Type 1 Gag Protein

The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.     

Regions of Human Immunodeficiency Virus Type 1 nef Required for Function In Vivo

In vivo studies in monkeys and humans have indicated that immunodeficiency viruses with Nef deleted are nonpathogenic in immunocompetent hosts, and this has motivated a search for live attenuated vaccine candidates. However, the mechanisms of action of Nef remain elusive. To define the regions of human immunodeficiency virus type 1 (HIV-1) Nef which mediate in vivo pathogenicity, a series of mutated isogenic viruses were inoculated into human thymic implants in SCID-hu mice. Mutation of several regions, including the myristoylation site at the second glycine and a region encompassing amino acids 41 through 49 of Nef, profoundly affected pathogenicity. Surprisingly, mutations of prolines in either of the two distant PXXP SH3 binding domains did not affect pathogenicity, indicating that these regions are not required for Nef activity in developing T-lineage cells. These data suggest that some functions of Nef described in vitro may not be relevant for in vivo pathogenicity.     

Role of a Putative gp41 Dimerization Domain in Human Immunodeficiency Virus Type 1 Membrane Fusion

The entry of human immunodeficiency virus type 1 (HIV-1) into a target cell entails a series of conformational changes in the gp41 transmembrane glycoprotein that mediates the fusion of the viral and target cell membranes. A trimer-of-hairpins structure formed by the association of two heptad repeat (HR) regions of the gp41 ectodomain has been implicated in a late step of the fusion pathway. Earlier native and intermediate states of the protein are postulated to mediate the antiviral activity of the fusion inhibitor enfuvirtide and of broadly neutralizing monoclonal antibodies (NAbs), but the details of these structures remain unknown. Here, we report the identification and crystal structure of a dimerization domain in the C-terminal ectodomain of gp41 (residues 630 to 683, or C54). Two C54 monomers associate to form an asymmetric, antiparallel coiled coil with two distinct C-terminal α-helical overhangs. This dimer structure is conferred largely by interactions within a central core that corresponds to the sequence of enfuvirtide. The mutagenic alteration of the dimer interface severely impairs the infectivity of Env-pseudotyped viruses. Moreover, the C54 structure binds tightly to both the 2F5 and 4E10 NAbs and likely represents a potential intermediate conformation of gp41. These results should enhance our understanding of the molecular basis of the gp41 fusogenic structural transitions and thereby guide rational, structure-based efforts to design new fusion inhibitors and vaccine candidates intended to induce broadly neutralizing antibodies.The entry of human immunodeficiency virus type 1 (HIV-1) into its target cell to establish an infection requires the fusion of viral and cellular membranes, a process that is mediated by the viral envelope glycoprotein (Env) through interactions with receptors on the target cell membrane (CD4 and a coreceptor, such as CCR-5 or CXCR-4) (14). HIV-1 Env is synthesized as the glycoprotein precursor gp160, which oligomerizes in the endoplasmic reticulum and subsequently is cleaved by the cellular furin endoprotease to create a metastable state that is primed for the induction of membrane fusion activity (19). The resulting Env complex is a trimeric structure comprising three gp120 surface glycoproteins, each associated noncovalently with one of three subunits of the gp41 transmembrane glycoprotein (24, 27, 47, 48). This native (prefusion) Env spike protrudes from the virus surface and is the target for neutralizing antibodies (NAbs) (reviewed in reference 3). It is generally accepted that HIV-1 membrane fusion is promoted by a series of receptor binding-triggered conformational changes in the Env complex, culminating in the formation of an energetically stable trimer of α-helical hairpins in gp41 (10, 14).The core structure of the trimer-of-hairpins is an antiparallel six-helix bundle: a central, three-stranded coiled coil formed by the first heptad repeat (HR_N) region of gp41 is sheathed by three α-helices derived from the second HR (HR_C) (5, 27, 42, 44). HR_N is immediately C terminal to the fusion peptide, while HR_C is adjacent to the transmembrane helix anchored in the viral membrane. The interaction of gp120 with CD4 and a chemokine receptor is thought to alter intersubunit interactions in the native Env complex, leading to gp41 reorganization into a postulated prehairpin intermediate (reviewed in references 10 and 14). At this point, the N-terminal HR_N coiled-coil trimer is formed, relocating the fusion peptides to allow them to insert into the cellular membrane. The HR_C region then is thought to jackknife so as to pack against the inner coiled-coil core and form the postfusion trimer-of-hairpin structure that brings the attached target cell and viral membranes together. Evidence for the existence of these different gp41 conformational states in the fusion pathway is indirect, being inferred from the antiviral activity of peptides derived from the two HR regions of gp41 (20, 45). These peptide inhibitors likely act in a dominant-negative manner by binding to the prehairpin intermediate, preventing the formation of the trimer-of-hairpins (6, 13, 27, 31). This intermediate is relatively stable, with a half-life of many minutes, as detected by the capacity of such peptides to inhibit fusion once prefusion gp41 has undergone a conformational transition (21, 31). Although mounting evidence indicates that the prefusogenic and intermediate states are important targets for drug- and vaccine-elicited NAbs (reviewed in references 3 and 10), little is known about their structures and how they modulate gp41 fusogenicity or serve as targets for inhibition.The C-terminal part of the gp41 ectodomain consists of HR_C (or C34) and the membrane-proximal external region (MPER) (Fig. ​(Fig.1).1). The C34 peptide is intrinsically disordered in isolation and forms an outer-layer α-helix only in the six-helix bundle (27, 29). Structural studies of the trimeric coiled-coil state of the MPER and of its bent helix conformation after binding to lipid membranes have begun to provide clues regarding the function of this unusual and important NAb-associated segment (25, 41). The MPER is the established target for two very rare but broadly reactive NAbs, 2F5 and 4E10/z13, which are elicited during natural human infection (50). These neutralizing epitopes seem to be poorly exposed on the surface of both HIV-1-infected cells and virions (reviewed in reference 3). Their exposure is enhanced or triggered by receptor binding but diminishes on the formation of the trimer-of-hairpins, suggesting that both of the NAbs target a more extended intermediate conformation rather than the native gp41 structure (8, 12). Despite extensive efforts, how structural aspects of the MPER explain its antigenicity and immunogenicity remains unclear. Here, we report the identification of the C-terminal dimerization domain of gp41 and present the 1.65-Å crystal structure of this domain. We characterize the role of this antiparallel two-stranded coiled-coil structure in NAb reactivity and viral function. Our study provides a potential structure for the fusion-intermediate state of gp41 and for the future design of new HIV-1 immunogens that may elicit broad and potent NAbs.Open in a separate windowFIG. 1.Structural and functional domains of HIV-1 gp41. (Upper) Schematic view of gp41 showing the location of the fusion peptide (FP), the two HR regions, the MPER, the transmembrane segment (TM), and the cytoplasmic region (CP). HR_C and MPER are depicted in blue and green, respectively. (Lower) Sequences of the C56, C54, C54_N656L, and C39 peptides employed in the study. The Asn-656→Leu mutation in C54_N656L is shown in red. The sequences of T-20 and core epitopes recognized by the human 2F5 and 4E10 MAbs are indicated.     

Ubiquitin Is Covalently Attached to the p6Gag Proteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12Gag Protein of Moloney Murine Leukemia Virus

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6^Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12^Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6^Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.     

A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4⁺ T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4⁺ T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.     

Cleavage of Human Immunodeficiency Virus Type 1 Proteinase from the N-Terminally Adjacent p6* Protein Is Essential for Efficient Gag Polyprotein Processing and Viral Infectivity

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.     

Actin Associates with the Nucleocapsid Domain of the Human Immunodeficiency Virus Gag Polyprotein

Recently, it was shown that actin molecules are present in human immunodeficiency virus type 1 (HIV-1) particles. We have examined the basis for incorporation and the location of actin molecules within HIV-1 and murine retrovirus particles. Our results show that the retroviral Gag polyprotein is sufficient for actin uptake. Immunolabeling studies demonstrate that actin molecules localize to a specific radial position within the immature particle, clearly displaced from the matrix domain underneath the viral membrane but in proximity to the nucleocapsid (NC) domain of the Gag polyprotein. When virus or subviral Gag particles were disrupted with nonionic detergent, actin molecules remained associated with the disrupted particles. Actin molecules remained in a stable complex with the NC cleavage product (or an NC-RNA complex) after treatment of the disrupted HIV-1 particles with recombinant HIV-1 protease. In contrast, matrix and capsid molecules were released. The same result was obtained when mature HIV-1 particles were disrupted with detergent. Taken together, these results indicate that actin molecules are associated with the NC domain of the viral polyprotein.     

Molecular Determinants in tRNA D-arm Required for Inhibition of HIV-1 Gag Membrane Binding

Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P₂ and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P₂-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNA^Pro, but not that of tRNA^Lys3, are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNA^Lys3 showed greater inhibition of MA membrane binding than full-length tRNA^Pro. While transplantation of the D loop sequence of tRNA^Lys3 into tRNA^Pro resulted in a modest increase in the inhibitory effect relative to WT tRNA^Pro, replacing the entire D arm sequence with that of tRNA^Lys3 was necessary to confer the full inhibitory effects upon tRNA^Pro. Together, these results demonstrate that the D arm of tRNA^Lys3 is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.