ABSTRACT
The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Sulfolobaceae/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Binding Sites , Gene Expression Regulation, Archaeal/physiology , HIV-1/physiology , Models, Molecular , Mutation , Protein Conformation , Sulfolobaceae/geneticsABSTRACT
HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.
Subject(s)
HIV-1/chemistry , HIV-1/physiology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cell Line , Down-Regulation , Gene Deletion , Gene Products, gag/metabolism , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/drug effects , HIV-1/growth & development , Host-Pathogen Interactions/drug effects , Humans , Leukemia Virus, Murine/chemistry , Membrane Glycoproteins , Membrane Proteins/deficiency , Membrane Proteins/pharmacology , Neoplasm Proteins/deficiency , Neoplasm Proteins/pharmacology , Protein Transport , Receptors, Cell Surface/deficiency , Virion/chemistry , Virion/drug effects , Virion/growth & development , Virion/physiology , Virus Replication/drug effects , nef Gene Products, Human Immunodeficiency Virus/deficiencyABSTRACT
UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the µ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis. IMPORTANCE: The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.
Subject(s)
Adaptor Protein Complex 2/metabolism , Adaptor Protein Complex mu Subunits/metabolism , Gene Products, gag/metabolism , HIV Infections/metabolism , HIV Infections/virology , HIV-1/physiology , Moloney murine leukemia virus/metabolism , Adaptor Protein Complex 2/genetics , Adaptor Protein Complex mu Subunits/genetics , Animals , Gene Products, gag/chemistry , Gene Products, gag/genetics , Gene Products, nef/chemistry , Gene Products, nef/genetics , Gene Products, nef/metabolism , Glycosylation , HIV Infections/genetics , HIV-1/genetics , Humans , Mice , Moloney murine leukemia virus/chemistry , Moloney murine leukemia virus/genetics , Protein Structure, Tertiary , nef Gene Products, Human Immunodeficiency Virus/deficiency , nef Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The SF1 helicase MOV10 is an antiviral factor that is incorporated into human immunodeficiency virus type 1 (HIV-1) virions. We now report that HIV-1 virions also incorporate UPF1, which belongs to the same SF1 helicase subfamily as MOV10 and functions in the nonsense-mediated decay (NMD) pathway. Unlike ectopic MOV10, the overexpression of UPF1 does not impair the infectivity of HIV-1 progeny virions. However, UPF1 becomes a potent inhibitor of HIV-1 progeny virion infectivity when residues required for its helicase activity are mutated. In contrast, equivalent mutations abolish the antiviral activity of MOV10. Importantly, cells depleted of endogenous UPF1, but not of another NMD core component, produce HIV-1 virions of substantially lower specific infectivity. The defect is at the level of reverse transcription, the same stage of the HIV-1 life cycle inhibited by ectopic MOV10. Thus, whereas ectopic MOV10 restricts HIV-1 replication, the related UPF1 helicase functions as a cofactor at an early postentry step.
Subject(s)
Host-Pathogen Interactions , Trans-Activators/metabolism , Virus Replication , HIV-1/physiology , Humans , Mutant Proteins/genetics , Mutant Proteins/metabolism , RNA Helicases , Trans-Activators/geneticsABSTRACT
HIV-1 employs its structural proteins to orchestrate assembly and budding at the plasma membrane of host cells, which depends on numerous cellular factors. Although cells evolved interferon inducible restriction factors such as tetherin that act as a first line of defense, enveloped viruses, including HIV-1, developed countermeasures in the form of tetherin antagonists such as Vpu that decrease the effect of tetherin and permits normal viral replication in vivo. Here we review recent advances in the understanding of the dynamic structural properties of tetherin that provide the basis to physically retain HIV-1 by bridging plasma and virion membranes after completion of budding.
Subject(s)
Antigens, CD/genetics , HIV-1/immunology , Mutation , Viral Regulatory and Accessory Proteins/metabolism , Antigens, CD/ultrastructure , Cell Line , Female , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/genetics , GPI-Linked Proteins/ultrastructure , HIV-1/pathogenicity , Host-Pathogen Interactions , Human Immunodeficiency Virus Proteins/metabolism , Humans , Male , Virus ReplicationABSTRACT
The detachment of human immunodeficiency type 1 (HIV-1) virions depends on CHPM4 family members, which are late-acting components of the ESCRT pathway that mediate the cleavage of bud necks from the cytosolic side. We now show that in human cells, CHMP4 proteins are to a considerable extent bound to two high-molecular-weight proteins that we have identified as CC2D1A and CC2D1B. Both proteins bind to the core domain of CHMP4B, which has a strong propensity to polymerize and to inhibit HIV-1 budding. Further mapping showed that CC2D1A binds to an N-terminal hairpin within the CHMP4 core that has been implicated in polymerization. Consistent with a model in which CC2D1A and CC2D1B regulate CHMP4 polymerization, the overexpression of CC2D1A inhibited both the release of wild-type HIV-1 and the CHMP4-dependent rescue of an HIV-1 L domain mutant by exogenous ALIX. Furthermore, small interfering RNA against CC2D1A or CC2D1B increased HIV-1 budding under certain conditions. CC2D1A and CC2D1B possess four Drosophila melanogaster 14 (DM14) domains, and we demonstrate that these constitute novel CHMP4 binding modules. The DM14 domain that bound most avidly to CHMP4B was by itself sufficient to inhibit the function of ALIX in HIV-1 budding, indicating that the inhibition occurred through CHMP4 sequestration. However, N-terminal fragments of CC2D1A that did not interact with CHMP4B nevertheless retained a significant level of inhibitory activity. Thus, CC2D1A may also affect HIV-1 budding in a CHMP4-independent manner.
Subject(s)
DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , HIV Infections/metabolism , HIV-1/physiology , Virus Release , Cell Line , DNA-Binding Proteins/genetics , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/genetics , HIV Infections/genetics , HIV Infections/virology , HIV-1/genetics , Humans , Protein Binding , Protein Structure, Tertiary , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
The Gag-Pol polyprotein of human immunodeficiency virus type 1 (HIV-1) is not required for efficient viral particle production. However, premature termination codons in pol, particularly in the integrase (IN)-coding region, can markedly impair HIV-1 particle formation, apparently due to the premature activation of the viral protease (PR). We now report that the IN domain of Gag-Pol is required for the incorporation of clathrin into HIV-1 virions. Significantly, PR-dependent effects of point mutations in IN on particle production correlated strictly with their effects on clathrin incorporation. A possible interpretation of these findings is that certain IN mutations impair particle production in a PR-dependent manner by promoting Gag-Pol dimerization, which also occludes a binding site for clathrin. Consistently with this model, the reverse transcriptase (RT) inhibitor efavirenz, which is thought to promote Gag-Pol dimerization, inhibited the incorporation of clathrin into HIV-1 virions. Clathrin-depleted cells produced normal amounts of HIV-1 virions; however, their infectivity was reduced. We also observed that HIV-2 and the simian immunodeficiency virus SIVmac interact with clathrin through one or two copies of a peptide motif in the p6 domain of Gag that resembles the clathrin box of cellular adaptor proteins. Furthermore, the substitution of the hydrophobic residues in the single clathrin box motif of SIVmac caused a replication defect in primary cells. Taken together, our results indicate that primate lentiviruses from two different subgroups functionally interact with clathrin during assembly.
Subject(s)
Clathrin/metabolism , HIV-1/physiology , Virus Assembly , gag Gene Products, Human Immunodeficiency Virus/metabolism , pol Gene Products, Human Immunodeficiency Virus/metabolism , Animals , HIV-2/physiology , Humans , Lentiviruses, Primate , Protein Binding , Simian Immunodeficiency Virus/physiology , Virion/chemistryABSTRACT
Many enveloped viruses depend on the membrane remodeling machinery of their host cells to complete their life cycle. In this issue, Rossman et al. (2010) now demonstrate that influenza virus possesses its own device for releasing nascent virus particles from the plasma membrane, the M2 proton-selective ion channel, which can substitute for the host cell's ESCRT pathway.
ABSTRACT
To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z(WT)) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z(WT) became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , HIV-1/physiology , Host-Pathogen Interactions , Nucleocapsid/metabolism , Virus Release , gag Gene Products, Human Immunodeficiency Virus/metabolism , Cell Line , Humans , Microscopy, Electron, Transmission , Nucleocapsid/genetics , Protein Interaction Domains and Motifs , Recombination, Genetic , Sequence Deletion , gag Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
ALIX recruits ESCRT-III CHMP4 and is involved in membrane remodeling during endosomal receptor sorting, budding of some enveloped viruses, and cytokinesis. We show that ALIX dimerizes via the middle domain (ALIX(-V)) in solution. Structural modeling based on small angle X-ray scattering (SAXS) data reveals an elongated crescent-shaped conformation for dimeric ALIX lacking the proline-rich domain (ALIX(BRO1-V)). Mutations at the dimerization interface prevent dimerization and induce an open elongated monomeric conformation of ALIX(-V) as determined by SAXS modeling. ALIX dimerizes in vivo and dimeric ALIX colocalizes with CHMP4B upon coexpression. We show further that ALIX dimerization affects HIV-1 budding. C-terminally truncated activated CHMP4B retaining the ALIX binding site forms linear, circular, and helical filaments in vitro, which can be bridged by ALIX. Our data suggest that dimeric ALIX represents the active form that interacts with ESCRT-III CHMP4 polymers and functions as a scaffolding protein during membrane remodeling processes.
Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Endosomes/virology , Binding Sites , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/ultrastructure , Carrier Proteins/ultrastructure , Cell Cycle Proteins/genetics , Cell Cycle Proteins/ultrastructure , Cell Line , Dimerization , Endosomal Sorting Complexes Required for Transport , Endosomes/metabolism , Genetic Complementation Test , HIV-1/physiology , Kidney/cytology , Models, Molecular , Molecular Weight , Mutation , Protein Binding , Protein Conformation , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
To promote the release of infectious virions, human immunodeficiency virus type 1 (HIV-1) exploits the endosomal sorting complex required for transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in p6 Gag. An LYPx(n)L motif in p6 serves as docking site for the central V domain of ALIX and is required for its ability to stimulate HIV-1 budding. Additionally, the nucleocapsid (NC) domain of Gag binds to the N-terminal Bro1 domain of ALIX, which connects ALIX to the membrane-deforming ESCRT-III complex via its CHMP4 subunits. Since the isolated Bro1 domain of ALIX is sufficient to markedly stimulate virus-like particle (VLP) production in a minimal Gag rescue assay, we examined whether the Bro1 domains of other human proteins possess a similar activity. We now show that the Bro1 domain-only protein Brox and the isolated Bro1 domains of HD-PTP and rhophilin all bind to HIV-1 NC. Furthermore, all shared the capacity to stimulate VLP production by a minimal HIV-1 Gag molecule, and Brox in particular was as potent as the Bro1 domain of ALIX in this assay. Unexpectedly, Brox retained significant activity even if its CHMP4 binding site was disrupted. Thus, the ability to assist in VLP production may be an intrinsic property of the boomerang-shaped Bro1 domain.
Subject(s)
HIV Infections/metabolism , HIV-1/physiology , Virion/physiology , gag Gene Products, Human Immunodeficiency Virus/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Endosomal Sorting Complexes Required for Transport , HIV Infections/virology , HIV-1/genetics , Humans , Protein Binding , Protein Structure, Tertiary , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , Virus Assembly , gag Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
ESCRT-III (endosomal sorting complex required for transport III) is required for the formation and abscission of intraluminal endosomal vesicles, which gives rise to multivesicular bodies, budding of some enveloped viruses and cytokinesis. ESCRT-III is composed of 11 members in humans, which, except for one, correspond to the six ESCRT-III-like proteins in yeast. At least CHMP (charged multivesicular body protein) 2A and CHMP3 assemble into helical tubular structures that provide a platform for membrane interaction and VPS (vacuolar protein sorting) 4-catalysed effects leading to disassembly of ESCRT-III CHMP2A-CHMP3 polymers in vitro. Progress towards the understanding of the structures and function of ESCRT-III, its activation, its regulation by accessory factors and its role in abscission of membrane enveloped structures in concert with VPS4 are discussed.
Subject(s)
Endosomes/chemistry , Endosomes/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Animals , Biopolymers/metabolism , Humans , Protein Binding , Protein TransportABSTRACT
Nef is an accessory protein of human immunodeficiency virus type 1 (HIV-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells. The requirement for Nef for optimal infectivity is, at least in part, determined by the envelope (Env) glycoprotein, because it can be eliminated by pseudotyping HIV-1 particles with pH-dependent Env proteins. To investigate the role of Env in the function of Nef, we have examined the effect of Nef on the infectivity of Env-deficient HIV-1 particles pseudotyped with viral receptors for cells expressing cognate Env proteins. We found that Nef significantly enhances the infectivity of CD4-chemokine receptor pseudotypes for cells expressing HIV-1 Env. Nef also increased the infectivity of HIV-1 particles pseudotyped with Tva, the receptor for subgroup A Rous sarcoma virus (RSV-A), even though Nef had no effect if the pH-dependent Env protein of RSV-A was used for pseudotyping. However, Nef does not always enhance viral infectivity if the normal orientation of the Env-receptor interaction is reversed, because the entry of Env-deficient HIV-1 into cells expressing the vesicular stomatitis virus G protein was unaffected by Nef. Together, our results demonstrate that the presence of a viral Env protein during virus production is not required for the ability of Nef to increase viral infectivity. Furthermore, since the infectivity of Tva pseudotypes was blocked by inhibitors of endosomal acidification, we conclude that low-pH-dependent entry does not always bypass the requirement for Nef.
Subject(s)
CD4 Antigens/metabolism , HIV-1/physiology , Virus Internalization , env Gene Products, Human Immunodeficiency Virus/metabolism , nef Gene Products, Human Immunodeficiency Virus/physiology , Avian Proteins/genetics , Avian Proteins/metabolism , CD4 Antigens/genetics , HIV-1/genetics , HIV-1/growth & development , Humans , Hydrogen-Ion Concentration , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , env Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs (endosomal sorting complexes required for transport) interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies; for the budding of some enveloped viruses; and for daughter cell scission in cytokinesis. We found that the ESCRT-III proteins CHMP2A and CHMP3 (charged multivesicular body proteins 2A and 3) could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule, whereas the AAA-type adenosine triphosphatase VPS4 could bind on the inside of the tubule and disassemble the tubes upon adenosine triphosphate hydrolysis. CHMP2A and CHMP3 copolymerized in solution, and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly budding vesicle, catalyzing late steps in budding under the control of VPS4.
Subject(s)
Adenosine Triphosphatases/metabolism , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/metabolism , Vesicular Transport Proteins/metabolism , Adenosine Triphosphate/metabolism , Centrifugation, Density Gradient , Dimerization , Endosomal Sorting Complexes Required for Transport , Humans , Lipid Bilayers/chemistry , Models, Molecular , Protein Conformation , Protein Structure, Secondary , Unilamellar Liposomes/chemistry , Vesicular Transport Proteins/chemistryABSTRACT
Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family--cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.
Subject(s)
Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Calorimetry , Circular Dichroism , Models, Molecular , Nerve Tissue Proteins/genetics , Protein Binding , Protein Structure, Tertiary , Ubiquitin Thiolesterase/chemistry , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolismABSTRACT
To exit infected cells, human immunodeficiency virus type 1 (HIV-1) exploits the vacuolar protein-sorting pathway by engaging Tsg101 and ALIX through PTAP and LYPx(n)L late assembly (L) domains. In contrast, less-complex retroviruses often use PPxY L domains to recruit Nedd4 family ubiquitin ligases. Although HIV-1 Gag lacks PPxY motifs, we now show that the budding of various HIV-1 L-domain mutants is dramatically enhanced by ectopic Nedd4-2s, a native isoform with a truncated C2 domain. The effect of Nedd4-2s on HIV-1 budding required a catalytically active HECT domain and was specific, since other Nedd4 family proteins showed little activity and an unrelated retrovirus was not rescued. The residual C2 domain of Nedd4-2s was critical for the enhancement of HIV-1 budding and for the association of Nedd4-2s with Gag, as reflected by its incorporation into virus-like particles. Interestingly, the incorporation of Nedd4-2s also depended on its active site, indicating that the ability to form a thioester with ubiquitin was required. These data suggest a novel mechanism by which HIV-1 Gag can connect to cellular budding machinery.
Subject(s)
HIV-1/growth & development , Ubiquitin-Protein Ligases/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , Amino Acid Motifs , Cell Line , Endosomal Sorting Complexes Required for Transport , Humans , Nedd4 Ubiquitin Protein Ligases , Protein Binding , Protein Interaction Mapping , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , gag Gene Products, Human Immunodeficiency Virus/geneticsSubject(s)
Antigens, CD/metabolism , HIV Infections/metabolism , HIV Infections/virology , HIV-1/metabolism , Human Immunodeficiency Virus Proteins/metabolism , Membrane Glycoproteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Animals , Antigens, CD/chemistry , Cell Membrane/virology , GPI-Linked Proteins , HIV Infections/therapy , HIV-1/drug effects , Herpesvirus 8, Human/enzymology , Herpesvirus 8, Human/physiology , Human Immunodeficiency Virus Proteins/deficiency , Human Immunodeficiency Virus Proteins/genetics , Humans , Interferon-alpha/pharmacology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/deficiency , Models, Biological , Ubiquitin-Protein Ligases/metabolism , Viral Regulatory and Accessory Proteins/deficiency , Viral Regulatory and Accessory Proteins/geneticsABSTRACT
Human immunodeficiency virus type 1 (HIV-1) and other retroviruses harbor short peptide motifs in Gag that promote the release of infectious virions. These motifs, known as late assembly (L) domains, recruit a cellular budding machinery that is required for the formation of multivesicular bodies (MVBs). The primary L domain of HIV-1 maps to a PTAP motif in the p6 region of Gag and engages the MVB pathway by binding to Tsg101. Additionally, HIV-1 p6 harbors an auxiliary L domain that binds to the V domain of ALIX, another component of the MVB pathway. We now show that ALIX also binds to the nucleocapsid (NC) domain of HIV-1 Gag and that ALIX and its isolated Bro1 domain can be specifically packaged into viral particles via NC. The interaction with ALIX depended on the zinc fingers of NC, which mediate the specific packaging of genomic viral RNA, but was not disrupted by nuclease treatment. We also observed that HIV-1 zinc finger mutants were defective for particle production and exhibited a similar defect in Gag processing as a PTAP deletion mutant. The effects of the zinc finger and PTAP mutations were not additive, suggesting a functional relationship between NC and p6. However, in contrast to the PTAP deletion mutant, the double mutants could not be rescued by overexpressing ALIX, further supporting the notion that NC plays a role in virus release.
Subject(s)
Calcium-Binding Proteins/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , HIV-1/physiology , gag Gene Products, Human Immunodeficiency Virus/metabolism , Endosomal Sorting Complexes Required for Transport , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , gag Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. We now show that dynamin 2 (Dyn2), a key regulator of vesicular trafficking, is a binding partner of Nef that is required for its ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by small interfering RNA potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells, this function of Nef could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The infectivity enhancement by Nef also depended on clathrin, because it was diminished in clathrin-depleted cells and profoundly inhibited in cells expressing the clathrin-binding domain of AP180, which blocks clathrin-coated pit formation but not clathrin-independent endocytosis. Together, these findings imply that the infectivity enhancement activity of Nef depends on Dyn2- and clathrin-mediated membrane invagination events.
Subject(s)
Dynamin II/physiology , Gene Products, nef/physiology , HIV-1/pathogenicity , Animals , COS Cells , Cell Line , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Gene Products, nef/deficiency , Gene Products, nef/genetics , HIV Infections/metabolism , HIV-1/physiology , Humans , Jurkat Cells , Protein Binding/physiology , Virion/pathogenicity , Virion/physiology , nef Gene Products, Human Immunodeficiency VirusABSTRACT
The release of human immunodeficiency virus type 1 (HIV-1) and of other retroviruses from certain cells requires the presence of distinct regions in Gag that have been termed late assembly (L) domains. HIV-1 harbors a PTAP-type L domain in the p6 region of Gag that engages an endosomal budding machinery through Tsg101. In addition, an auxiliary L domain near the C terminus of p6 binds to ALIX/AIP1, which functions in the same endosomal sorting pathway as Tsg101. In the present study, we show that the profound release defect of HIV-1 L domain mutants can be completely rescued by increasing the cellular expression levels of ALIX and that this rescue depends on an intact ALIX binding site in p6. Furthermore, the ability of ALIX to rescue viral budding in this system depended on two putative surface-exposed hydrophobic patches on its N-terminal Bro1 domain. One of these patches mediates the interaction between ALIX and the ESCRT-III component CHMP4B, and mutations which disrupt the interaction also abolish the activity of ALIX in viral budding. The ability of ALIX to rescue a PTAP mutant also depends on its C-terminal proline-rich domain (PRD), but not on the binding sites for Tsg101, endophilin, CIN85, or for the newly identified binding partner, CMS, within the PRD. Our data establish that ALIX can have a dramatic effect on HIV-1 release and suggest that the ability to use ALIX may allow HIV-1 to replicate in cells that express only low levels of Tsg101.