The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.
Newly formed HIV-1 particles assemble at the plasma membrane of virus producing cells. The inner structural protein Gag and the envelope glycoprotein Env, which are both essential components of infectious virus particles, traffic to the membrane via different pathways. Attached to the inner side of the membrane, Gag assembles into spherical particles that incorporate Env proteins in their surrounding lipid envelope. The mechanism of Env incorporation is incompletely understood, however. Here, we have exploited recently developed super-resolution fluorescence microscopy techniques that yield a near-molecular spatial resolution to analyze HIV-1 Gag and Env distribution patterns at the surface of virus producing cells. We observed recruitment of Env to the surroundings of Gag assembly sites, dependent on the presence of its cytoplasmic domain. A large proportion of Env was found in the vicinity of the Gag assembly sites rather than directly co-localizing with it. These results support an indirect mechanism of Env recruitment, presumably mediated through virus induced changes in the environment of the nascent Gag assembly. Furthermore, they suggest a role for the Env protein in HIV-1 transmission that goes beyond its well-characterized function as an entry protein on the viral surface.
Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.
Entry of human immunodeficiency virus type 1 (HIV-1) commences with binding of the envelope glycoprotein (Env) to the receptor CD4, and one of two coreceptors, CXCR4 or CCR5. Env-mediated signaling through coreceptor results in Gαq-mediated Rac activation and actin cytoskeleton rearrangements necessary for fusion. Guanine nucleotide exchange factors (GEFs) activate Rac and regulate its downstream protein effectors. In this study we show that Env-induced Rac activation is mediated by the Rac GEF Tiam-1, which associates with the adaptor protein IRSp53 to link Rac to the Wave2 complex. Rac and the tyrosine kinase Abl then activate the Wave2 complex and promote Arp2/3-dependent actin polymerization. Env-mediated cell-cell fusion, virus-cell fusion and HIV-1 infection are dependent on Tiam-1, Abl, IRSp53, Wave2, and Arp3 as shown by attenuation of fusion and infection in cells expressing siRNA targeted to these signaling components. HIV-1 Env-dependent cell-cell fusion, virus-cell fusion and infection were also inhibited by Abl kinase inhibitors, imatinib, nilotinib, and dasatinib. Treatment of cells with Abl kinase inhibitors did not affect cell viability or surface expression of CD4 and CCR5. Similar results with inhibitors and siRNAs were obtained when Env-dependent cell-cell fusion, virus-cell fusion or infection was measured, and when cell lines or primary cells were the target. Using membrane curving agents and fluorescence microscopy, we showed that inhibition of Abl kinase activity arrests fusion at the hemifusion (lipid mixing) step, suggesting a role for Abl-mediated actin remodeling in pore formation and expansion. These results suggest a potential utility of Abl kinase inhibitors to treat HIV-1 infected patients.
Patients infected with HIV-1 are currently treated with highly active antiretroviral therapy (HAART) that efficiently suppresses the virus but does not cure the infection. HIV-1 envelope activates Rac-mediated actin cytoskeleton rearrangements in the target cell that promote membrane fusion and entry. We discovered that these rearrangements require activation of the actin polymerization machinery including the tyrosine kinase Abl. We also showed that Abl kinase inhibitors imatinib, nilotinib, and dasatinib, current drug therapies for chronic myeloid leukemia, block HIV-1 entry and infection. These results suggest that these inhibitors might be appropriate drugs for treatment of HIV-1. This strategy of using inhibitors that disable host signaling proteins rather than viral proteins, essential for pathogen survival, may have a general efficacy in developing drugs to combat HIV-1 and other pathogens that acquire drug resistance.
We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR+) or inactive (PR−) viral PR. We observed that PR− virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.
Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.
Attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. Influenza A virus (IAV) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. IAV subsequently enters cells of birds and a wide variety of mammals via receptor-mediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). The elucidation of the endocytic pathways taken by IAV has been hampered by their apparent redundancy in establishing a productive infection. By manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of IAV. Collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative IAV entry route. As the dynamin-dependent and -independent IAV entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative IAV entry route. A similar strategy might be applicable to the study of endocytic pathways taken by other viruses.
The interferon-inducible transmembrane protein BST-2 (CD317, tetherin) restricts the release of several enveloped viruses from infected cells. BST-2 is broadly active against retroviruses, including HIV-1 and HIV-2. To counteract this host defense, HIV-1 uses the accessory protein Vpu, whereas HIV-2 uses its envelope glycoprotein (Env). In both cases, viral antagonism is associated with decreased expression of BST-2 at the cell surface. Here, we provide evidence supporting a role for the clathrin-mediated endocytic pathway in the downregulation of BST-2 from the cell surface and the counteraction of restricted virion release. A catalytically inactive, dominant negative version of the vesicle “pinch-ase” dynamin 2 (dyn2K44A) inhibited the downregulation of BST-2 by Vpu, and it inhibited the release of wild-type (Vpu-expressing) HIV-1 virions. Similarly, dyn2K44A inhibited the downregulation of BST-2 by HIV-2 Env, and it inhibited the release of vpu-negative HIV-1 virions when HIV-2 Env was provided in trans. dyn2K44A inhibited Env more robustly than Vpu, suggesting that dynamin 2, while a cofactor for both Env and Vpu, might support just one of several pathways though which Vpu counteracts BST-2. In support of a role for clathrin in these effects, the C-terminal domain of the clathrin assembly protein AP180 also inhibited the downregulation of BST-2 by either Vpu or HIV-2 Env. Consistent with modulation of the postendocytic itinerary of BST-2, Vpu enhanced the accumulation of cell surface-derived BST-2 in transferrin-containing endosomes. Vpu also inhibited the transport of BST-2 from a brefeldin A-insensitive compartment to the cell surface, consistent with a block to endosomal recycling. We propose that HIV-1 Vpu, and probably HIV-2 Env, traps BST-2 in an endosomal compartment following endocytosis, reducing its level at the cell surface to counteract restricted viral release.
We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.
We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear.
We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization.
Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.
HIV fusion kinetics; hemifusion; fusion pore; dynasore; temperature-arrested intermediate; fusion inhibitors; single particle tracking; beta-lactamase; intrinsic membrane curvature
The matrix (MA) domain of HIV-1 Gag plays key roles in membrane targeting of Gag, and envelope (Env) glycoprotein incorporation into virions. Although a trimeric MA structure has been available since 1996, evidence for functional MA trimers has been elusive. The mechanism of HIV-1 Env recruitment into virions likewise remains unclear. Here, we identify a point mutation in MA that rescues the Env incorporation defects imposed by an extensive panel of MA and Env mutations. Mapping the mutations onto the putative MA trimer reveals that the incorporation-defective mutations cluster at the tips of the trimer, around the perimeter of a putative gap in the MA lattice into which the cytoplasmic tail of gp41 could insert. By contrast, the rescue mutation is located at the trimer interface, suggesting that it may confer rescue of Env incorporation via modification of MA trimer interactions, a hypothesis consistent with additional mutational analysis. These data strongly support the existence of MA trimers in the immature Gag lattice and demonstrate that rescue of Env incorporation defects is mediated by modified interactions at the MA trimer interface. The data support the hypothesis that mutations in MA that block Env incorporation do so by imposing a steric clash with the gp41 cytoplasmic tail, rather than by disrupting a specific MA-gp41 interaction. The importance of the trimer interface in rescuing Env incorporation suggests that the trimeric arrangement of MA may be a critical factor in permitting incorporation of Env into the Gag lattice.
One of the enduring problems in HIV-1 research is the mechanism of incorporation of the viral envelope (Env) glycoprotein into viral particles. Several models have been proposed ranging from an entirely passive process to a requirement for binding of Env by the matrix (MA) domain of the Gag precursor polyprotein. It is clear that specific regions within MA and Env play important roles, as mutations in these domains can prevent Env incorporation. We have identified a point mutation in MA that rescues a broad range of Env-incorporation defective mutations, located both in MA and in Env. Our investigations into the mechanism of rescue have revealed the importance of interactions between MA monomers at a trimeric interface. Our results are consistent with previously published crystallographic models and now provide functional support for the existence of MA trimers in the immature Gag lattice. Furthermore, as the modification of trimer interactions plays a role in the rescue of Env incorporation, we propose that MA trimerization and the organization of the MA lattice may be critical factors in Env incorporation.
The Env glycoproteins of retroviruses play an important role in the initial steps of infection involving the binding to cell surface receptors and entry by membrane fusion. The Env glycoprotein also plays an important role in viral assembly at a late step of infection. Although the Env glycoprotein interacts with viral matrix proteins and cellular proteins associated with lipid rafts, its possible role during the early replication events remains unclear. Truncation of the cytoplasmic tail (CT) of the Env glycoprotein is acquired by SIV in the course of adaptation to human cells, and is known to be a determinant of SIV pathogenicity.
We compared SIV viruses with full length or truncated (T) Env glycoproteins to analyze possible differences in entry and post-entry events, and assembly of virions. We observed that early steps in replication of SIV with full length or T Env were similar in dividing and non-dividing cells. However, the proviral DNA of the pathogenic virus clone SIVmac239 with full length Env was imported to the nucleus about 20-fold more efficiently than proviral DNA of SIVmac239T with T Env, and 100-fold more efficiently than an SIVmac18T variant with a single mutation A239T in the SU subunit and with a truncated cytoplasmic tail (CT). In contrast, proviral DNA of SIVmac18 with a full length CT and with a single mutation A239T in the SU subunit was imported to the nucleus about 50-fold more efficiently than SIVmac18T. SIV particles with full length Env were released from rhesus monkey PBMC, whereas a restriction of release of virus particles was observed from human 293T, CEMx174, HUT78 or macrophages. In contrast, SIV with T Envs were able to overcome the inhibition of release in human HUT78, CEMx174, 293T or growth-arrested CEMx174 cells and macrophages resulting in production of infectious particles. We found that the long CT of the Env glycoprotein was required for association of Env with lipid rafts. An Env mutant C787S which eliminated palmitoylation did not abolish Env incorporation into lipid rafts, but prevented virus assembly.
The results indicate that the long cytoplasmic tail of the SIV Env glycoprotein may govern post-entry replication events and plays a role in the assembly process.
The mechanisms involved in the incorporation of viral glycoproteins into virions are incompletely understood. For retroviruses, incorporation may involve interactions between the Gag proteins of these viruses and the cytoplasmic domains of the relevant envelope (Env) glycoproteins. Recent studies have identified within the cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) Env protein a tyrosine-containing internalization motif similar to those found in the cytoplasmic domains of certain cell surface proteins that undergo rapid constitutive endocytosis in clathrin-coated pits. Given that surface expression of the HIV-1 Env protein is essential for the production of infectious virus, the presence of this internalization motif is surprising. We show here that in contrast to the rapid rate of Env protein internalization observed in cells expressing the Env protein in the absence of other HIV-1 proteins, the rate of internalization of Env protein from the surfaces of HIV-1-infected cells is extremely slow. The presence of the Pr55gag precursor protein is necessary and sufficient for inhibition of Env protein internalization, while a mutant Pr55-gag that is incapable of mediating Env incorporation into virions is also unable to inhibit endocytosis of the Env protein. The failure of the Env protein to undergo endocytosis from the surface of an HIV-1-infected cell may reflect the fact that the proposed interaction of the matrix domain of the Gag protein with Env during assembly prevents the interaction of Env with host adaptin molecules that recruit plasma membrane molecules such as the transferrin receptor into clathrin-coated pits. When the normal ratio of Gag and Env proteins in the infected cells is altered by overexpression of Env protein, this mechanism allows removal of excess Env protein from the cell surface. Taken together, these results suggest that a highly conserved system to reduce surface levels of the Env protein functions to remove Env protein that is not associated with Gag and that is therefore not destined for incorporation into virions. This mechanism for the regulation of surface levels of Env protein may protect infected cells from Env-dependent cytopathic effects or Env-specific immune responses.
HIV-1 entry into cells is a multifaceted process involving target cell CD4 and the chemokine receptors, CXCR4 or CCR5. The lipid composition of the host cell plays a significant role in the HIV fusion process as it orchestrates the appropriate disposition of CD4 and co-receptors required for HIV-1 envelope glycoprotein (Env)-mediated fusion. The cell membrane is primarily composed of sphingolipids and cholesterol. The effects of lipid modulation on CD4 disposition in the membrane and their role in HIV-1 entry have extensively been studied. To focus on the role of lipid composition on chemokine receptor function, we have by-passed the CD4 requirement for HIV-1 Env-mediated fusion by using a CD4-independent strain of HIV-1 Env.
Cell fusion mediated by a CD4-independent strain of HIV-1 Env was monitored by observing dye transfer between Env-expressing cells and NIH3T3 cells bearing CXCR4 or CCR5 in the presence or absence of CD4. Chemokine receptor signaling was assessed by monitoring changes in intracellular [Ca2+] mobilization induced by CCR5 or CXCR4 ligand. To modulate target membrane cholesterol or sphingolipids we used Methyl-β-cyclodextrin (MβCD) or 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), respectively. Treatment of the target cells with these agents did not change the levels of CD4 or CXCR4, but reduced levels of CCR5 on the cell surface. Chemokine receptor signalling was inhibited by cholesterol removal but not by treatment with PPMP. HIV-1 Env mediated fusion was inhibited by >50% by cholesterol removal. Overall, PPMP treatment appeared to slow down the rates of CD4-independent HIV-1 Env-mediated Fusion. However, in the case of CXCR4-dependent fusion, the differences between untreated and PPMP-treated cells did not appear to be significant.
Although modulation of cholesterol and sphingolipids has similar effects on CD4 -dependent HIV-1 Env-mediated fusion, sphingolipid modulation had little effect on CD4-independent HIV-1 Env-mediated fusion. Chemokine receptor function remained intact following treatment of cells with PPMP. Therefore such treatment may be considered a more suitable agent to inhibit CD4 dependent HIV-1 infection.
Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits and the stability of the Env complex.
A novel entry mechanism has been proposed for the avian sarcoma and leukosis virus (ASLV), whereby interaction with specific cell surface receptors activates or primes the viral envelope glycoprotein (Env), rendering it sensitive to subsequent low-pH-dependent fusion triggering in acidic intracellular organelles. However, ASLV fusion seems to proceed to a lipid mixing stage at neutral pH, leading to the suggestion that low pH might instead be required for a later stage of viral entry such as uncoating (L. J. Earp, S. E. Delos, R. C. Netter, P. Bates, and J. M. White. J. Virol. 77:3058-3066, 2003). To address this possibility, hybrid virus particles were generated with the core of human immunodeficiency virus type 1 (HIV-1), a known pH-independent virus, and with subgroups A or B ASLV Env proteins. Infection of cells by these pseudotyped virions was blocked by lysosomotropic agents, as judged by inhibition of HIV-1 DNA synthesis. Furthermore, by using HIV-1 cores that contain a Vpr-β-lactamase fusion protein (Vpr-BlaM) to monitor viral penetration into the cytosol, we demonstrated that virions bearing ASLV Env, but not HIV-1 Env, enter the cytosol in a low-pH-dependent manner. This effect was independent of the presence of the cytoplasmic tail of ASLV Env. These studies provide strong support for the model, indicating that low pH is required for ASLV Env-dependent viral penetration into the cytosol and not for viral uncoating.
Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry.
Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T cells through adhesive contacts called virological synapses (VSs). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1-neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry-based infection assays to measure the inhibitory potency of HIV-1-neutralizing monoclonal antibodies (MAb) and HIV-1-neutralizing patient sera against cell-free and VS-mediated infection. To various degrees, anti-Env MAbs exhibited significantly higher 50% inhibitory concentration (IC50s) against VS-mediated infection than cell-free infection. Notably, the MAb 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inocula compared to cell-free inocula. A mutant with truncation mutation in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to virus particle maturation but which can still engage in cell-to-cell infection was tested for the ability to resist neutralizing antibodies. The ΔCT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread.
The gp41 component of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) contains a long cytoplasmic domain (CD) with multiple highly conserved tyrosine (Y) and dileucine (LL) motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL), located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed.
All mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.
From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.
The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor, gp160, that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and co-receptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation, and the role of specific membrane microdomains in this process. Here we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions.
Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this “inside-out” regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.
The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates. To date, no effective therapies have been identified. To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope. Fusion was detected by cleavage of the fluorogenic substrate CCF2 by β-lactamase-Vpr incorporated into virions and released as a result of virion fusion. Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1. These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion. Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol). Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion. Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs. In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion. We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells. Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis. Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.
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.
HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.
HIV infection occurs following the binding of viral envelope glycoproteins (Env) to receptors on target cell surfaces. Binding to these molecules induces conformational changes in Env, ultimately leading to the exposure of a viral fusion peptide and fusion of viral and cellular membranes. Understanding the structure of Env at each step during HIV entry is of fundamental importance in the design of compounds that can combat infection. Here, we use cryo-electron tomography to characterize the conformational changes that occur in Env at individual steps in the entry process, revealing the unexpected finding that binding to a co-receptor mimic alone induces the same conformational changes as binding to CD4. Furthermore, using single particle cryo-electron microscopy, we show structural evidence, at sub-nanometer resolution, of a novel, activated intermediate state of HIV where highly conserved, interior components of the viral spike are exposed. We show that transition to this state can be blocked by addition of a highly neutralizing antibody, VRC01, revealing a possible mechanism for its potent neutralizing ability. Discovery of the structure of this new Env intermediate provides a template for the design of immunogens aimed at eliciting antibodies that could block HIV entry.
The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (ΔV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.
The envelope protein of HIV-1 is responsible for binding virus to the surface of cells and mediating viral entry. Viral entry can be prevented by neutralizing antibodies that bind to envelope, and by small molecule inhibitors that bind to viral receptors on the cell surface, such as CCR5. HIV may acquire resistance to these small molecule inhibitors, several of which are being used in clinical trials to treat HIV-infected individuals, through resistance mechanisms that are not well understood. In addition, broadly neutralizing antibodies are rare—the envelope protein possesses structural features that limit antibody binding. We made a partial deletion in a region of envelope that interacts with viral receptors, and which is also widely believed to act as a shield against neutralizing antibodies. Normally, an envelope with such a modification would have total loss of function. However, by passaging virus with the partially deleted envelope in vitro, the envelope acquired adaptive mutations that restored function. Virus with the adapted envelope was highly sensitive to neutralizing antibodies and so may serve as a platform for immunization. This envelope also exhibited complete resistance to small molecule inhibitors that bind to the viral receptor CCR5, and lends insight into a mechanism of drug resistance by which the virus interacts with viral receptors on the cell surface in a novel manner.
The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a “snorkeling” model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.
HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescence of the HIV gp41 N-helical and C-helical regions, which results in the formation of 6 helix bundles. Kinetics of HIV Env-mediated fusion is an important determinant of sensitivity to entry inhibitors and antibodies. However, the parameters that govern the HIV Env fusion cascade have yet to be fully elucidated. We address this issue by comparing the kinetics HIV-1IIIB Env with those mediated by HIV-2 from two strains with different affinities for CD4 and CXCR4.
HIV-1 and HIV-2 Env-mediated cell fusion occurred with half times of about 60 and 30 min, respectively. Binding experiments of soluble HIV gp120 proteins to CD4 and co-receptor did not correlate with the differences in kinetics of fusion mediated by the three different HIV Envs. However, escape from inhibition by reagents that block gp120-CD4 binding, CD4-induced CXCR4 binding and 6-helix bundle formation, respectively, indicated large difference between HIV-1 and HIV-2 envelope glycoproteins in their CD4-induced rates of engagement with CXCR4.
The HIV-2 Env proteins studied here exhibited a significantly reduced window of time between the engagement of gp120 with CD4 and exposure of the CXCR4 binding site on gp120 as compared with HIV-1IIIB Env. The efficiency with which HIV-2 Env undergoes this CD4-induced conformational change is the major cause of the relatively rapid rate of HIV-2 Env mediated-fusion.