Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.     

An acidic cluster of the cytoplasmic tail of the RD114 virus glycoprotein controls assembly of retroviral envelopes

Retroviral core proteins, Gag and envelope (Env) glycoproteins are expressed from distinct cellular areas and therefore need to encounter to assemble infectious particles. The intrinsic cell localisation properties of either viral component or their capacity to mutually interact determines the assembly of infectious particles. Here, we address how Env determinants and cellular sorting proteins allow the Env derived from gamma retroviruses, murine leukemia virus (MLV) and RD114, to travel to or from late endosomes (LE), which may represent the Env assembly site of retroviruses in some cells. The individual expression of MLV Env resulted in its accumulation in LE in contrast to RD114 Env that required the presence of gamma retroviral Gag proteins. To discriminate between intrinsic intracellular Env localisation and gamma retroviral Gag/Env interactions in influencing Env viral incorporation, we studied Env assembly on heterologous lentiviral particles on which they are passively recruited. We found that an acidic cluster present at the C-terminus of the RD114 Env cytoplasmic tail determines its sub-cellular localisation and retrograde transport. Mutation of this motif induced late endosomal concentration of the RD114 Env, correlating with increased viral incorporation and infectivity. Reciprocally, the reinforcement of a poorly functional acidic motif in the MLV Env resulted in a marked decrease of its late endosomal localisation, leading to weakly infectious lentiviral particles with low Env densities. Finally, through upregulation versus downregulation of its cellular expression, we show that phosphofurin acidic-cluster-sorting protein 1 (PACS-1) controls the function of the RD114 Env acidic cluster, assigning to this cellular effector a crucial role in modulation of Env assembly of some retroviruses.     

Targeting of the human immunodeficiency virus type 1 envelope to the trans-Golgi network through binding to TIP47 is required for env incorporation into virions and infectivity

Here, we report that human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is located mainly in the trans-Golgi network (TGN) due to determinants present in the cytoplasmic domain of the transmembrane gp41 glycoprotein (TMgp41). Internalization assays demonstrated that Env present at the cell surface returns to the TGN. We found that the cytoplasmic domain of TMgp41 binds to TIP47, a protein required for the transport of mannose-6-phosphate receptors from endosomes to the TGN. Overexpression of a mutant of TIP47 affected the transport of Env from endosomes to the TGN. Retrograde transport of Env to the TGN requires a Y(802)W(803) diaromatic motif present in the TMgp41 cytoplasmic domain. Mutation of this motif abolished both targeting to the TGN as well as interaction with TIP47. These data support the view that binding of TIP47 to HIV-1 Env facilitates its delivery to the TGN. Lastly, we show that virus mutated in the Y(802)W(803) motif is poorly infectious and presents a defect in Env incorporation, supporting a model in which retrograde transport of Env is implicated in the optimization of fully infectious HIV-1 production.     

Human Discs Large is a new negative regulator of human immunodeficiency virus-1 infectivity

Human immunodeficiency virus (HIV)-1 replication is positively or negatively regulated through multiple interactions with host cell proteins. We report here that human Discs Large (Dlg1), a scaffold protein recruited beneath the plasma membrane and involved in the assembly of multiprotein complexes, restricts HIV-1 infectivity. The endogenous Dlg1 and HIV-1 Gag polyprotein spontaneously interact in HIV-1-chronically infected T cells. Depleting endogenous Dlg1 in either adherent cells or T cells does not affect Gag maturation, production, or release, but it enhances the infectivity of progeny viruses five- to sixfold. Conversely, overexpression of Dlg1 reduces virus infectivity by ~80%. Higher virus infectivity upon Dlg1 depletion correlates with increased Env content in cells and virions, whereas the amount of virus-associated Gag or genomic RNA remains identical. Dlg1 knockdown is also associated with the redistribution and colocalization of Gag and Env toward CD63 and CD82 positive vesicle-like structures, including structures that seem to still be connected to the plasma membrane. This study identifies both a new negative regulator that targets the very late steps of the HIV-1 life cycle, and an assembly pathway that optimizes HIV-1 infectivity.     

HIV-1 assembly: viral glycoproteins segregate quantally to lipid rafts that associate individually with HIV-1 capsids and virions

HIV-1 assembly depends on its structural protein, Gag, which after synthesis on ribosomes, traffics to the late endosome/plasma membrane, associates with HIV Env glycoprotein, and forms infectious virions. While Env and Gag migrate to lipid microdomains, their stoichiometry and specificity of interaction are unknown. Pseudotyped viral particles can be made with one viral core surrounded by heterologous envelope proteins. Taking advantage of this property, we analyzed the association of HIV Env and Ebola glycoprotein (GP), with HIV-1 Gag coexpressed in the same cell. Though both viral glycoproteins were expressed, each associated independently with Gag, giving rise to distinct virion populations, each with a single glycoprotein type. Confocal imaging demonstrated that Env and GP localized to distinct lipid raft microdomains within the same cell where they associated with different virions. Thus, a single Gag particle associates "quantally" with one lipid raft, containing homogeneous trimeric viral envelope proteins, to assemble functional virions.     

Positive and negative modulation of virus infectivity and envelope glycoprotein incorporation into virions by amino acid substitutions at the N terminus of the simian immunodeficiency virus matrix protein

The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal alpha-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within alpha-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the beta-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.     

The cellular protein lyric interacts with HIV-1 Gag

Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein driving assembly and release of virions from infected cells. Gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. In a series of affinity purification experiments, we identified the cellular protein Lyric to be an HIV-1 Gag-interacting protein. Lyric was previously described to be an HIV-inducible gene and is involved in various signaling pathways. Gag interacts with endogenous Lyric via its matrix (MA) and nucleocapsid (NC) domains. This interaction requires Gag multimerization and Lyric amino acids 101 to 289. Endogenous Lyric is incorporated into HIV-1 virions and is cleaved by the viral protease. Gag-Lyric interaction was also observed for murine leukemia virus and equine infectious anemia virus, suggesting that it represents a conserved feature among retroviruses. Expression of the Gag binding domain of Lyric increased Gag expression levels and viral infectivity, whereas expression of a Lyric mutant lacking the Gag binding site resulted in lower Gag expression and decreased viral infectivity. The results of the current study identify Lyric to be a cellular interaction partner of HIV-1 Gag and hint at a potential role in regulating infectivity. Further experiments are needed to elucidate the precise role of this interaction.     

Evidence that Vpu Modulates HIV-1 Gag-Envelope Interaction towards Envelope Incorporation and Infectivity in a Cell Type Dependent Manner

The HIV-1 Vpu is required for efficient virus particle release from the plasma membrane and intracellular CD4 degradation in infected cells. In the present study, we found that the loss of virus infectivity as a result of envelope (Env) incorporation defect caused by a Gag matrix (MA) mutation (L30E) was significantly alleviated by introducing a start codon mutation in vpu. Inactivation of Vpu partially restored the Env incorporation defect imposed by L30E substitution in MA. This effect was found to be comparable in cell types such as 293T, HeLa, NP2 and GHOST as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). However, in HeLa cells BST-2 knockdown was found to further alleviate the effect of Vpu inactivation on infectivity of L30E mutant. Our data demonstrated that the impaired infectivity of virus particles due to Env incorporation defect caused by MA mutation was modulated by start codon mutation in Vpu.     

The dominant-negative action of a fusion protein containing the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 in virus replication

We previously described a novel mode of downregulation of human immunodeficiency virus type 1 (HIV-1) Gag expression by a cytoplasmic domain fusion protein of the envelope (Env) transmembrane protein, β-galactosidase (β-gal)/706–856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli β-gal. In the present study, we showed that this mediator conferred a dose-dependent dominant interference with virus infectivity. In the context of an HIV-1 provirus, this inhibitor downregulated steady-state Env expression. Paradoxically, Env overexpression suppressed β-gal/706–856-mediatd Gag downregulation. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag, Env, and β-gal/706–856 had stable interactions and formed aggregated complexes in perinuclear regions. Moreover, Env overexpression hindered colocalization of Gag with β-gal/706–856 in the perinuclear region. Further cytoplasmic domain mapping analyses showed a correlation between the ability of cytoplasmic subdomains to downregulate Gag expression and the ability of these subdomains to stably interact with Gag. These studies show that redirection of Gag from its cytoplasmic synthesis site to a perinuclear compartment is a prerequisite for β-gal/706–856-mediated Gag downregulation. The results also illustrate that the dynamic interplay among Gag, Env, and β-gal/706–856 can modulate Gag and Env expression, thus controlling HIV-1 infection.     

Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.     

Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection.

The matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein contains a highly basic region near its amino terminus. It has been proposed that this basic domain, in conjunction with the HIV-1 accessory protein Vpr, is responsible for the localization of the HIV-1 preintegration complex to the nucleus in nondividing cells. It has also been postulated that the matrix basic domain assists in the targeting of the HIV-1 Gag precursor Pr55Gag to the plasma membrane during virus assembly. To evaluate the role of this highly basic sequence during infection of primary human monocyte-derived macrophages, single- and double-amino-acid-substitution mutations were introduced, and the effects on virus particle production, Gag protein processing, envelope glycoprotein incorporation into virus particles, and virus infectivity in the CEM(12D-7) T-cell line, peripheral blood mononuclear cells, and primary human monocyte-derived macrophages were analyzed. Although modest effects on virus particle production were observed with some of the mutants, none abolished infectivity in primary human monocyte-derived macrophages. In contrast with previously reported studies involving some of the same matrix basic domain mutants, infectivity in monocyte-derived macrophages was retained even when combined with a vpr mutation.     

Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. By using gene trap insertional mutagenesis, we identified Rab9, which mediates late-endosome-to-trans-Golgi-network trafficking, among several candidate host genes whose disruption allowed the survival of Marburg virus-infected cells, suggesting that Rab9 is utilized in Marburg replication. Although Rab9 has not been implicated in human immunodeficiency virus (HIV) replication, previous reports suggested that the late endosome is an initiation site for HIV assembly and that TIP47-dependent trafficking out of the late endosome to the trans-Golgi network facilitates the sorting of HIV Env into virions budding at the plasma membrane. We examined the role of Rab9 in the life cycles of HIV and several unrelated viruses, using small interfering RNA (siRNA) to silence Rab9 expression before viral infection. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to-trans-Golgi vesicular transport. In addition, silencing studies revealed that HIV replication was dependent on the expression of Rab11A, which mediates trans-Golgi-to-plasma-membrane transport, and that increased HIV Gag was sequestered in a CD63+ endocytic compartment in a cell line stably expressing Rab9 siRNA. Replication of the enveloped Ebola, Marburg, and measles viruses was inhibited with Rab9 siRNA, although the non-enveloped reovirus was insensitive to Rab9 silencing. These results suggest that Rab9 is an important cellular target for inhibiting diverse viruses and help to define a late-endosome-to-plasma-membrane vesicular transport pathway important in viral assembly.     

HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores

Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.     

Insulin-like growth factor II mRNA binding protein 1 associates with Gag protein of human immunodeficiency virus type 1, and its overexpression affects virus assembly

The assembly of human immunodeficiency virus type 1 (HIV-1) particles is driven by viral Gag protein. This function of Gag not only benefits from its self-multimerization property but also depends on its interaction with a number of cellular factors such as TSG101 and ALIX/AIP1 that promote virus budding and release from cell surfaces. However, interaction with Gag also allows some cellular factors such as APOBEC3G and Trim5alpha to access viral replication machinery and block viral replication. In this study, we report a new HIV-1 Gag-binding factor named insulin-like growth factor II mRNA binding protein 1 (IMP1). Gag-IMP1 interaction requires the second zinc finger of the nucleocapsid (NC) domain of Gag and the KH3 and KH4 domains of IMP1. A fourfold reduction of HIV-1 infectivity was seen with overexpression of the wild-type IMP1 and its mutant that is able to interact with Gag but not with overexpression of IMP1 mutants exhibiting Gag-binding deficiency. The decreased viral infectivity was further shown as a result of diminished viral RNA packaging, abrogated Gag processing on the cellular membranes, and impeded maturation of virus particles. Together, these results demonstrate that IMP1 interacts with HIV-1 Gag protein and is able to block the formation of infectious HIV-1 particles.     

Characterization of prototype foamy virus gag late assembly domain motifs and their role in particle egress and infectivity

Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101.     

Comparative immunogenicity of human immunodeficiency virus particles and corresponding polypeptides in a DNA vaccine

The immunogenicity of a plasmid DNA expression vector encoding both Gag and envelope (Env), which produced human immunodeficiency virus (HIV) type 1 virus-like particles (VLP), was compared to vectors expressing Gag and Env individually, which presented the same gene products as polypeptides. Vaccination with plasmids that generated VLP showed cellular immunity comparable to that of Gag and cell-mediated or humoral responses similar to those of Env as immunization with separate vectors. These data suggest that DNA vaccines encoding separated HIV polypeptides generate immune responses similar to those generated by viral particles.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.     

A highly conserved residue in the C-terminal helix of HIV-1 matrix is required for envelope incorporation into virus particles

The incorporation of viral envelope (Env) glycoproteins into nascent particles is an essential step in the production of infectious human immunodeficiency virus type 1 (HIV-1). This process has been shown to require interactions between Env and the matrix (MA) domain of the Gag polyprotein. Previous studies indicate that several residues in the N-terminal region of MA are required for Env incorporation. However, the precise mechanism by which Env proteins are acquired during virus assembly has yet to be fully defined. Here, we examine whether a highly conserved glutamate at position 99 in the C-terminal helix is required for MA function and HIV-1 replication. We analyze a panel of mutant viruses that contain different amino acid substitutions at this position using viral infectivity studies, virus-cell fusion assays, and immunoblotting. We find that E99V mutant viruses are defective for fusion with cell membranes and thus are noninfectious. We show that E99V mutant particles of HIV-1 strains LAI and NL4.3 lack wild-type levels of Env proteins. We identify a compensatory substitution in MA residue 84 and show that it can reverse the E99V-associated defects. Taken together, these results indicate that the C-terminal hydrophobic pocket of MA, which encompasses both residues 84 and 99, has a previously unsuspected and key role in HIV-1 Env incorporation.     

Utility of human immunodeficiency virus type 1 envelope as a T-cell immunogen

Human immunodeficiency virus (HIV)-specific CD8 T lymphocytes are important for the control of viremia, but the relative utility of responses to the various HIV proteins is controversial. Immune responses that force escape mutations that exact a significant fitness cost from the mutating virus would help slow progression to AIDS. The HIV envelope (Env) protein is subject to both humoral and cellular immune responses, suggesting that multiple rounds of mutation are needed to facilitate viral escape. The Gag protein, however, has recently been shown to elicit a more effective CD8 T-cell immune response in humans. We studied 30 pigtail macaques for their CD8 T-lymphocyte responses to HIV-1 Env and simian immunodeficiency virus (SIV) Gag following prime/boost vaccination and intrarectal challenge with simian-human immunodeficiency virus SHIV_mn229. Eight CD8 Env-specific T-cell epitopes were identified and mapped in 10 animals. Animals that generated Env-specific CD8 T-cell responses had equivalent viral loads and only a modest advantage in retention of peripheral CD4 T lymphocytes compared to those animals without responses to Env. This contrasts with animals that generated CD8 T-cell responses to SIV Gag in the same trial, demonstrating superior control of viral load and a larger advantage in retention of peripheral CD4 T cells than Gag nonresponders. Mutational escape was common in Env but, in contrast to mutations in Gag, did not result in the rapid emergence of dominant escape motifs, suggesting modest selective pressure from Env-specific T cells. These results suggest that Env may have limited utility as a CD8 T-cell immunogen.     

Effect of extension of the cytoplasmic domain of human immunodeficiency type 1 virus transmembrane protein gp41 on virus replication

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.