Effects of Gag mutation and processing on retroviral dimeric RNA maturation

After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.     

Hydrogen/deuterium exchange analysis of HIV-1 capsid assembly and maturation

Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in?vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated.     

Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents.

We have examined structural interactions of Gag proteins in human immunodeficiency virus type 1 (HIV-1) particles by utilizing cysteine mutagenesis and cysteine-specific modifying reagents. In immature protease-minus but otherwise wild-type (wt) particles, precursor Pr55Gag proteins did not form intermolecular cystines naturally but could be cross-linked at cysteines, and cross-linking appeared to occur across nucleocapsid (NC) domains. Capsid (CA) proteins in wt mature viruses possess cysteines near their carboxy termini at gag codons 330 and 350, but these residues are not involved in natural covalent intermolecular bonds, nor can they be intermolecularly cross-linked by using the membrane-permeable cross-linker bis-maleimido hexane. The cysteine at gag codon 350 (C-350) is highly reactive to thiol-specific modifying reagents, while the one at codon 330 (C-330) appears considerably less reactive, even in the presence of ionic detergent. These results suggest that the HIV-1 CA C terminus forms an unusually stable conformation. Mutagenesis of C-350 to a serine residue in the mutant C350S (C-350 changed to serine) virtually eliminated particle assembly, attesting to the importance of this region. We also examined a C330S mutant, as well as mutants in which cysteines were created midway through the capsid domain or in the C-terminal section of the major homology region. All such mutants appeared wt on the basis of biochemical assays but showed greatly reduced infectivities, indicative of a postassembly, postprocessing replicative block. Interestingly, capsid proteins of mature major homology region mutant particles could be cysteine cross-linked, implying either that these mutations permit cross-linking of the native C-terminal CA cysteines or that major homology regions on neighbor capsid proteins are in close proximity in mature virions.     

Human immunodeficiency virus type 1 resistance to the small molecule maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid is conferred by a variety of single amino acid substitutions at the CA-SP1 cleavage site in Gag

The compound 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.     

Maturation of dimeric viral RNA of Moloney murine leukemia virus.

We have analyzed the dimeric RNA present in Moloney murine leukemia virus (MoMuLV) particles. We found that the RNA in newly released virions is in a conformation different from that in mature virions, since it has a different electrophoretic mobility in nondenaturing agarose gels and dissociates into monomers at a lower temperature. On the basis of these results, we suggest that the RNA initially packaged into nascent virions is already dimeric but that the dimer undergoes a maturation process after the virus is released from the cell. In further experiments, we tested the possibility that this maturation event is linked to the maturation cleavage of the virion proteins, which is catalyzed by the viral protease (PR). We found that the dimeric RNA isolated from PR- mutant virions resembles that from immature virions: it has a lower electrophoretic mobility and a lower sedimentation rate, and it also dissociates at a lower temperature than does RNA from mature wild-type virions. When Kirsten sarcoma virus is rescued by a PR- mutant or by a somewhat leaky cysteine array mutant of MoMuLV, its RNA also exhibits a electrophoretic mobility lower than that in the wild-type pseudotype. These results suggest that the maturation of dimeric RNA in released virus particles requires the cleavage of the Gag precursor and the presence of an intact cysteine array in the released nucleocapsid protein.     

HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice

Maturation of nascent virions, a key step in retroviral replication, involves cleavage of the Gag polyprotein by the viral protease into its matrix (MA), capsid (CA), and nucleocapsid (NC) components and their subsequent reorganization. Bevirimat (BVM) defines a new class of antiviral drugs termed maturation inhibitors. BVM acts by blocking the final cleavage event in Gag processing, the separation of CA from its C-terminal spacer peptide 1 (SP1). Prior evidence suggests that BVM binds to Gag assembled in immature virions, preventing the protease from accessing the CA-SP1 cleavage site. To investigate this hypothesis, we used cryo-electron tomography to examine the structures of (noninfectious) HIV-1 viral particles isolated from BVM-treated cells. We find that these particles contain an incomplete shell of density underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost, NC-related, layer. We conclude that the shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVM-treated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no visible evidence of honeycomb organization, indicative of an altered conformation and further suggesting that binding of BVM stabilizes the immature lattice. In both cases, the observed failure to assemble mature capsids correlates with the loss of infectivity.     

Overexpression and incorporation of GagPol precursor does not impede packaging of HIV-1 tRNA<Superscript><Emphasis Type="Italic">Lys3</Emphasis></Superscript> but promotes intracellular budding of virus-like particles

We have recently demonstrated that alteration of the human immunodeficiency virus type 1 (HIV-1) Gag/Gag-Pol ratio in virus-producing cells reduces the infectivity of progeny viruses and hinders the formation of stable virion RNA dimers without impairing virion packaging of the viral genomic RNA. In addition, we have previously shown that the expression of GagPol mediates the selective packaging of tRNA ^Lys3 . In this study we report that overexpression of uncleaved GagPol in the virus-producing cell did not alter the packaging levels of tRNA ^Lys3 . Similarly, altering the virion-associated Gag/GagPol ratio did not affect the virion packaging of the HIV-1 envelope protein nor cyclophilin A. Thin section electron microscopy analysis of the cells overexpressing protease-defective [PR(-)] GagPol revealed immature virions but no mature virions. These immature virions were seen both extracellularly and in membrane-bound cytoplasmic vacuoles. Furthermore, an accumulation of electron-dense material was occasionally found at the plasma membrane and associated with intracytoplasmic membranous vacuoles in cells expressing excess PR(–) GagPol. No intracellular HIV was seen in the wild-type control. Density gradient analysis showed that the overall density of these mutant virions with excess PR(–) GagPol was identical to that of the wild-type HIV-1. The findings indicate that overexpression of PR(–) GagPol, in the presence of Gag synthesis, promotes intracellular budding of the mutant virions and inhibits virus maturation.     

The Effect of Inositol Hexakisphosphate on HIV-1 Particle Production and Infectivity can be Modulated by Mutations that Affect the Stability of the Immature Gag Lattice

The assembly of an HIV-1 particle begins with the construction of a spherical lattice composed of hexamer subunits of the Gag polyprotein. The cellular metabolite inositol hexakisphosphate (IP6) binds and stabilizes the immature Gag lattice via an interaction with the six-helix bundle (6HB), a crucial structural feature of Gag hexamers that modulates both virus assembly and infectivity. The 6HB must be stable enough to promote immature Gag lattice formation, but also flexible enough to be accessible to the viral protease, which cleaves the 6HB during particle maturation. 6HB cleavage liberates the capsid (CA) domain of Gag from the adjacent spacer peptide 1 (SP1) and IP6 from its binding site. This pool of IP6 molecules then promotes the assembly of CA into the mature conical capsid that is required for infection. Depletion of IP6 in virus-producer cells results in severe defects in assembly and infectivity of wild-type (WT) virions. Here we show that in an SP1 double mutant (M4L/T8I) with a hyperstable 6HB, IP6 can block virion infectivity by preventing CA-SP1 processing. Thus, depletion of IP6 in virus-producer cells markedly increases M4L/T8I CA-SP1 processing and infectivity. We also show that the introduction of the M4L/T8I mutations partially rescues the assembly and infectivity defects induced by IP6 depletion on WT virions, likely by increasing the affinity of the immature lattice for limiting IP6. These findings reinforce the importance of the 6HB in virus assembly, maturation, and infection and highlight the ability of IP6 to modulate 6HB stability.     

Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55(Gag) in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55(Gag) in HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55(Gag) in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55(Gag) at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55(Gag) and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55(Gag) with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.     

Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant

The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10(-6) relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.     

The role of the S-S bridge in retroviral protease function and virion maturation

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.     

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.     

The initial step in human immunodeficiency virus type 1 GagProPol processing can be regulated by reversible oxidation

Background

Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Methodology/Principal Findings

To determine if oxidation influences this step, we created a GagProPol plasmid construct (pGPfs-1C) that encoded mutations at all cleavage sites except p2/NC, the initial cleavage site in GagProPol. pGPfs-1C was used in an in vitro translation assay to observe the behavior of this initial step without interference from subsequent processing events. Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol. The ability to regulate processing by reversible oxidation was lost when the cysteines of the embedded protease were mutated to alanine. Unlike mature protease, which requires only oxidation of cys95 for inhibition, both cysteines of the embedded protease contributed to this inhibition.

Conclusions/Significance

We developed a system that can be used to study the first step in the cascade of HIV-1 GagProPol processing and show that reversible oxidation of cysteines of HIV-1 protease embedded in GagProPol can block this initial GagProPol autoprocessing. This type of regulation may be broadly applied to the majority of retroviruses.     

Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production

In primate cells, assembly of a single HIV-1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV-1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation. In the next approximately 2 h, Gag-ABCE1 complexes increase in size to approximately that of immature capsids. Dissociation of ABCE1 from Gag correlates closely with Gag processing during virion maturation and occurs much less efficiently when the HIV-1 protease is inactivated. Finally, quantitative double-label immunogold electron microscopy reveals that ABCE1 is recruited to sites of assembling wild-type Gag at the plasma membrane but not to sites of an assembly-defective Gag mutant at the plasma membrane. Together these findings demonstrate that a population of Gag present at plasma membrane sites of assembly associates with ABCE1 throughout capsid formation until the onset of virus maturation, which is then followed by virus release. Moreover, the data suggest a linkage between Gag-ABCE1 dissociation and subsequent events of virion production.     

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC.     

Cysteine residues in the Vif protein of human immunodeficiency virus type 1 are essential for viral infectivity.

The infectivity factor of human immunodeficiency virus type 1 (HIV-1), Vif, contains two cysteine residues which are highly conserved among animal lentiviruses. We introduced substitutions of leucine for cysteine residues in the vif gene of a full-length HIV-1 clone to analyze their roles in viral infection. Mutant viruses containing substitutions in either Cys-114, Cys-133, or both displayed a vif-negative infection phenotype similar to that of an isogeneic vif deletion mutant, namely, a cell-dependent complete to partial loss of infectivity. The vif defect could be complemented by cotransfection of mutant viral DNA with a Vif expression vector, and there was no evidence that recombination contributed to the repair of the vif deficiency. The viral protein profile, as determined by immunoblotting, in cells infected with cysteine substitution mutants and that in wild-type virus were similar, including the presence of the 23-kDa Vif polypeptide. In addition, immunoblotting with an antiserum directed against the carboxyl terminus of gp41 revealed that gp41 was intact in cells infected with either wild-type or vif mutant HIV-1, excluding that Vif cleaves the C terminus of gp41. Our results indicate that the cysteines in HIV-1 Vif are critical for Vif function in viral infectivity.     

Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity.

The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolytic activity prevents maturation, the level of activity sufficient for maturation and subsequent infectivity has not been determined. Amino acid substitutions that reduce catalytic activity without affecting substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles.     

Proline residues in human immunodeficiency virus type 1 p6(Gag) exert a cell type-dependent effect on viral replication and virion incorporation of Pol proteins

The C terminus of the HIV-1 Gag protein contains a proline-rich domain termed p6(Gag). This domain has been shown to play a role in efficient virus release and incorporation of Vpr into virions. In a previous study (X. F. Yu, L. Dawson, C. J. Tian, C. Flexner, and M. Dettenhofer, J. Virol. 72:3412-3417, 1998), we observed that the removal of the p6 domain of Gag as well as drastic mutations in the PTAP motif resulted in reduced virion-associated Pol proteins from transfected COS cells. In the present study, amino acid substitutions at residues 5 and 7 of p6(Gag) resulted in a cell type-dependent replication of the mutant virus in CD4(+) T cells; the virus was replication competent in Jurkat cells but restricted in H9 cells and primary blood-derived monocytes. Established Jurkat and H9 cell lines expressing p6(Gag) mutant and parental virus were used to further understand this defect. Mutant virions produced from H9 cells, which displayed no defect in extracellular virion production, showed an approximately 16-fold reduction in Pol protein levels, whereas the levels of Pol proteins were only marginally reduced in mutant virions produced from Jurkat cells. The reduction in the virion-associated Pol proteins could not be accounted for by differences in the levels of intracellular p160(Gag-Pol) or in the interaction between p55(Gag) and p160(Gag-Pol) precursors. Electron microscopic analysis of the p6(Gag) mutant virions showed a predominately immature morphology in the absence of significant defects in Gag proteolytic cleavage. Taken together, these data suggest that the proline-rich motif of p6(Gag) is involved in the late stages of virus maturation, which include the packaging of cleaved Pol proteins in viral particles, a process which may involve cell-type-specific factors.     

Maturation-dependent human immunodeficiency virus type 1 particle fusion requires a carboxyl-terminal region of the gp41 cytoplasmic tail

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode fusion glycoproteins with long cytoplasmic tails (CTs). We previously reported that immature HIV-1 particles are inhibited for fusion with target cells by a mechanism requiring the 152-amino-acid CT of gp41. The gp41 CT was also shown to mediate the detergent-resistant association of the HIV-1 envelope glycoprotein complex with immature HIV-1 particles, indicating that the gp41 CT forms a stable complex with Gag in immature virions. In the present study, we analyzed the effects of progressive truncations and point mutations in the gp41 CT on the fusion of mature and immature HIV-1 particles with target cells. We also determined the effects of these mutations on the detergent-resistant association of gp41 with immature HIV-1 particles. Removal of the C-terminal 28 amino acids relieved the dependence of HIV-1 fusion on maturation. However, a mutant Env protein lacking this region remained associated with immature HIV-1 particles treated with nonionic detergent. Further mutational analysis of the C-terminal region of gp41 revealed two specific sequences required for maturation-dependent HIV-1 fusion. Collectively, our results demonstrate that the extreme C terminus of gp41 plays a key role in coupling HIV-1 fusion competence to virion maturation. They further indicate that the stable association of gp41 with Gag in immature virions is not sufficient for inhibition of immature HIV-1 particle fusion.