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1.
mBio ; 14(1): e0338222, 2023 02 28.
Article in English | MEDLINE | ID: mdl-36622146

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.


Subject(s)
HIV-1 , Humans , Antiviral Agents/pharmacology , CD4 Antigens , Cell Line , Membrane Glycoproteins , Membrane Proteins/metabolism , nef Gene Products, Human Immunodeficiency Virus/genetics , Virus Replication
2.
Sci Adv ; 7(44): eabj7398, 2021 Oct 29.
Article in English | MEDLINE | ID: mdl-34714669

ABSTRACT

BST2 is an interferon-inducible antiviral host protein antagonized by HIV-1 Vpu that entraps nascent HIV-1 virions on the cell surface. Unexpectedly, we find that HIV-1 lacking Nef can revert to full replication competence simply by losing the ability to antagonize BST2. Using gene editing together with cell sorting, we demonstrate that even the propagation of wild-type HIV-1 is strikingly dependent on BST2, including in primary human cells. HIV-1 propagation in BST2−/− populations can be fully rescued by exogenous BST2 irrespective of its capacity to signal and even by an artificial BST2-like protein that shares its virion entrapment activity but lacks sequence homology. Counterintuitively, our results reveal that HIV-1 propagation is critically dependent on basal levels of virion tethering by a key component of innate antiviral immunity.

3.
Cell Rep ; 22(4): 869-875, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29386131

ABSTRACT

We recently identified the multipass transmembrane protein SERINC5 as an antiviral protein that can potently inhibit HIV-1 infectivity and is counteracted by HIV-1 Nef. We now report that the anti-HIV-1 activity, but not the sensitivity to Nef, is conserved among vertebrate SERINC5 proteins. However, a Nef-resistant SERINC5 became Nef sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. Our results establish that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef.


Subject(s)
Cytoplasm/metabolism , HIV-1/genetics , Membrane Proteins/genetics , nef Gene Products, Human Immunodeficiency Virus/genetics , Humans
4.
Nat Immunol ; 17(12): 1447-1458, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27798619

ABSTRACT

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.


Subject(s)
Germinal Center/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Orthomyxoviridae Infections/immunology , Th1 Cells/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Cells, Cultured , Humans , Immunoglobulin G/blood , Interferon-gamma/metabolism , Interleukins/genetics , Interleukins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout
5.
Nature ; 526(7572): 218-23, 2015 Oct 08.
Article in English | MEDLINE | ID: mdl-26416733

ABSTRACT

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/deficiency
6.
J Virol ; 88(6): 3443-54, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24403584

ABSTRACT

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/genetics
7.
Cell Rep ; 5(3): 802-12, 2013 Nov 14.
Article in English | MEDLINE | ID: mdl-24209751

ABSTRACT

HIV-1 Nef and the unrelated murine leukemia virus glycoGag similarly enhance the infectivity of HIV-1 virions. We now show that the effects of Nef and glycoGag are similarly determined by variable regions of HIV-1 gp120 that control Env trimer association and neutralization sensitivity. Whereas neutralization-sensitive X4-tropic Env proteins conferred high responsiveness to Nef and glycoGag, particles bearing neutralization-resistant R5-tropic Envs were considerably less affected. The profoundly different Nef/glycoGag responsiveness of a neutralization-resistant and a neutralization-sensitive R5-tropic Env could be switched by exchanging their gp120 V1/V2 regions, which also switches their neutralization sensitivity. Within V1/V2, the same determinants governed Nef/glycoGag responsiveness and neutralization sensitivity, indicating that these phenotypes are mechanistically linked. The V1/V2 and V3 regions, which form an apical trimer-association domain, together determined the Nef and glycoGag responsiveness of an X4-tropic Env. Our results suggest that Nef and glycoGag counteract the inactivation of Env spikes with relatively unstable apical trimer-association domains.


Subject(s)
HIV Envelope Protein gp120/genetics , HIV Infections/virology , HIV-1/genetics , env Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/genetics , Alleles , Amino Acid Sequence , HEK293 Cells , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp120/metabolism , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1/immunology , HIV-1/metabolism , Humans , Molecular Sequence Data , Transfection , env Gene Products, Human Immunodeficiency Virus/immunology , nef Gene Products, Human Immunodeficiency Virus/immunology , nef Gene Products, Human Immunodeficiency Virus/metabolism
8.
Curr HIV Res ; 10(4): 298-306, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22524178

ABSTRACT

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 Replication
9.
J Mol Biol ; 419(1-2): 75-88, 2012 May 25.
Article in English | MEDLINE | ID: mdl-22406677

ABSTRACT

Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/metabolism , Binding Sites , Cell Line, Transformed , DNA-Binding Proteins/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Endosomes/genetics , Endosomes/metabolism , HEK293 Cells , HIV-1/metabolism , Humans , Models, Molecular , Mutation/genetics , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolism
10.
J Virol ; 86(7): 3746-56, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22258254

ABSTRACT

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/metabolism
11.
Structure ; 19(8): 1149-59, 2011 Aug 10.
Article in English | MEDLINE | ID: mdl-21827950

ABSTRACT

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.


Subject(s)
Endosomal Sorting Complexes Required for Transport/chemistry , Multiprotein Complexes/chemistry , Peptide Fragments/chemistry , Ubiquitin Thiolesterase/chemistry , Amino Acid Sequence , Endosomal Sorting Complexes Required for Transport/metabolism , HEK293 Cells , HIV Infections/virology , HIV-1/physiology , Humans , Hydrogen Bonding , Molecular Sequence Data , Multiprotein Complexes/metabolism , Peptide Fragments/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Ubiquitin Thiolesterase/metabolism , Virus Release
12.
Cell Host Microbe ; 7(4): 314-323, 2010 Apr 22.
Article in English | MEDLINE | ID: mdl-20399176

ABSTRACT

The restriction factor BST-2/tetherin contains two membrane anchors employed to retain some enveloped viruses, including HIV-1 tethered to the plasma membrane in the absence of virus-encoded antagonists. The 2.77 A crystal structure of the BST-2/tetherin extracellular core presented here reveals a parallel 90 A long disulfide-linked coiled-coil domain, while the complete extracellular domain forms an extended 170 A long rod-like structure based on small-angle X-ray scattering data. Mutagenesis analyses indicate that both the coiled coil and the N-terminal region are required for retention of HIV-1, suggesting that the elongated structure can function as a molecular ruler to bridge long distances. The structure reveals substantial irregularities and instabilities throughout the coiled coil, which contribute to its low stability in the absence of disulfide bonds. We propose that the irregular coiled coil provides conformational flexibility, ensuring that BST-2/tetherin anchoring both in the plasma membrane and in the newly formed virus membrane is maintained during virus budding.


Subject(s)
Antigens, CD/chemistry , Cell Membrane/virology , HIV-1/physiology , Host-Pathogen Interactions , Membrane Glycoproteins/chemistry , Virus Release , Animals , Antigens, CD/metabolism , Circular Dichroism , Crystallography, X-Ray , GPI-Linked Proteins , Membrane Glycoproteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Scattering, Small Angle
13.
Biochem Soc Trans ; 37(Pt 1): 181-4, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19143627

ABSTRACT

HIV-1 Gag engages components of the ESCRT (endosomal sorting complex required for transport) pathway via so-called L (late-assembly) domains to promote virus budding. Specifically, the PTAP (Pro-Thr-Ala-Pro)-type primary L domain of HIV-1 recruits ESCRT-I by binding to Tsg101 (tumour susceptibility gene 101), and an auxiliary LYPX(n)L (Leu-Tyr-Pro-Xaa(n)-Leu)-type L domain recruits the ESCRT-III-binding partner Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X]. The structurally related CHMPs (charged multivesicular body proteins), which form ESCRT-III, are kept in an inactive state through intramolecular interactions, and become potent inhibitors of HIV-1 budding upon removal of an autoinhibitory region. In the absence of the primary L domain, HIV-1 budding is strongly impaired, but can be efficiently rescued through the overexpression of Alix. This effect of Alix depends on its ability to interact with CHMP4, suggesting that it is the recruitment of CHMPs that ultimately drives virus release. Surprisingly, HIV-1 budding defects can also be efficiently corrected by overexpressing Nedd (neural-precursor-cell-expressed developmentally down-regulated) 4-2s, a member of a family of ubiquitin ligases previously implicated in the function of PPXY (Pro-Pro-Xaa-Tyr)-type L domains, which are absent from HIV-1. At least under certain circumstances, Nedd4-2s stimulates the activity of PTAP-type L domains, raising the possibility that the ubiquitin ligase regulates the activity of ESCRT-I.


Subject(s)
Endosomes/metabolism , HIV-1/physiology , Multiprotein Complexes/metabolism , Endosomal Sorting Complexes Required for Transport , Endosomes/enzymology , Humans , Models, Biological , Multiprotein Complexes/chemistry , Nedd4 Ubiquitin Protein Ligases , Protein Binding , Protein Transport , Ubiquitin-Protein Ligases/metabolism , Vesicular Transport Proteins/metabolism
14.
J Virol ; 82(10): 4898-907, 2008 May.
Article in English | MEDLINE | ID: mdl-18321969

ABSTRACT

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/genetics
15.
J Virol ; 81(12): 6614-22, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17428861

ABSTRACT

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.


Subject(s)
Calcium-Binding Proteins/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , HIV-1/genetics , Transcription Factors/metabolism , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Binding Sites , Blotting, Western , Cell Line , Endosomal Sorting Complexes Required for Transport , Genetic Techniques , Humans , Mutation , Protein Binding , Protein Structure, Tertiary
16.
Proc Natl Acad Sci U S A ; 103(50): 19140-5, 2006 Dec 12.
Article in English | MEDLINE | ID: mdl-17146056

ABSTRACT

The endosomal sorting complex ESCRT-III, which is formed by the structurally related CHMP proteins, is engaged by HIV-1 to promote viral budding. Here we show that progressive truncations into the C-terminal acidic domains of CHMP proteins trigger an increasingly robust anti-HIV budding activity. Together with biochemical evidence for specific intramolecular interactions between the basic and acidic halves of CHMP3 and CHMP4B, these results suggest that the acidic domains are autoinhibitory. The acidic half of CHMP3 also interacts with the endosome-associated ubiquitin isopeptidase AMSH, and the coexpression of AMSH or its CHMP3-binding domain converts wild-type CHMP3 into a potent inhibitor of HIV-1 release. Point mutations in CHMP3 that prevent binding to AMSH abrogate this effect, suggesting that binding to AMSH relieves the autoinhibition of CHMP3. Collectively, our results indicate that CHMP proteins are regulated through an autoinhibitory switch mechanism that allows tight control of ESCRT-III assembly.


Subject(s)
HIV-1/physiology , Nerve Tissue Proteins/metabolism , Acids , Antiviral Agents/metabolism , Binding Sites , Cell Line , Endosomal Sorting Complexes Required for Transport , Humans , Mutation/genetics , Nerve Tissue Proteins/genetics , Protein Binding
17.
Dev Cell ; 10(6): 821-30, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16740483

ABSTRACT

The vacuolar protein sorting machinery regulates multivesicular body biogenesis and is selectively recruited by enveloped viruses to support budding. Here we report the crystal structure of the human ESCRT-III protein CHMP3 at 2.8 A resolution. The core structure of CHMP3 folds into a flat helical arrangement that assembles into a lattice, mainly via two different dimerization modes, and unilaterally exposes a highly basic surface. The C terminus, the target for Vps4-induced ESCRT disassembly, extends from the opposite side of the membrane targeting region. Mutations within the basic and dimerization regions hinder bilayer interaction in vivo and reverse the dominant-negative effect of a truncated CHMP3 fusion protein on HIV-1 budding. Thus, the final steps in the budding process may include CHMP protein polymerization and lattice formation on membranes by employing different bilayer-recognizing surfaces, a function shared by all CHMP family members.


Subject(s)
Crystallography, X-Ray , HIV Infections , HIV-1/physiology , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/metabolism , Amino Acid Sequence , Amino Acids, Acidic/chemistry , Amino Acids, Basic/chemistry , Conserved Sequence , Dimerization , Endosomal Sorting Complexes Required for Transport , HIV Infections/metabolism , HIV Infections/virology , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Structure, Secondary , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Static Electricity , Vesicular Transport Proteins/genetics , Virus Replication
18.
Biol Pharm Bull ; 27(2): 261-5, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14758049

ABSTRACT

The pharmacokinetic parameters of lopinavir (LPV) were examined by administering Kaletra (LPV+ritonavir) to 8 healthy Japanese volunteers both in the fasting and postprandial conditions. LPV showed a biphasic decline, which was slower in the initial phase and became more rapid in the later phase. The behavior of LPV in the initial phase could be modeled using a one-compartment model with first-order absorption. In the fasting study, calculations based on the pharmacokinetic model revealed that the time to reach the maximum concentration (T(max)), maximum concentration (C(max)), half-life (T(1/2)), lag time, apparent volume of distribution (Vd/F) and oral clearance (Cl/F) were 3.2+/-1.0 h, 6.9+/-1.9 microg/ml, 10.0+/-3.7 h, 0.71+/-0.32 h, 51.0+/-12.4 l and 4.2+/-2.6 l/h, respectively. On the other hand, in the postprandial study, the calculated T(max), C(max), T(1/2), lag time, Vd/F and Cl/F were 5.6+/-2.0 h, 7.6+/-1.8 microg/ml, 16.7+/-7.0 h, 2.35+/-0.78 h, 48.0+/-15.9 l and 2.1+/-0.6 l/h, respectively. The values for the area under the curve for data collected over a 24-h period (AUC(24 h)) in the fasting and postprandial studies were 86.0+/-27.7 and 102.1+/-31.0 microg.h/ml, respectively. The T(1/2) had a tendency to be prolonged after food intake, but there were 2 cases with shortened T(1/2). Food intake prolonged the lag time 3-fold and as a result, the postprandial T(max) was 2 times longer.


Subject(s)
Protease Inhibitors/pharmacokinetics , Pyrimidinones/pharmacokinetics , Ritonavir/pharmacokinetics , Administration, Oral , Adult , Drug Administration Schedule , Drug Combinations , Female , Food-Drug Interactions , Humans , Lopinavir , Male , Middle Aged , Models, Biological , Postprandial Period , Protease Inhibitors/blood , Pyrimidinones/blood , Time Factors
19.
Chem Pharm Bull (Tokyo) ; 51(6): 715-8, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12808252

ABSTRACT

We developed a simple HPLC method for the simultaneous determination of lopinavir (LPV), ritonavir (RTV) and efavirenz (EFV) to evaluate the efficiency of co-administration of LPV/RTV and EFV in Japanese patients enrolled in a clinical study. The monitoring of LPV plasma concentration is important because co-administration of LPV/RTV with EFV sometimes decreases plasma concentrations of LPV caused by EFV activation of cytochrome P-450 3A. A solution of acetonitrile, methanol and tetramethylammonium perchlorate (TMAP) in dilute aqueous trifluoroacetic acid (TFA) has been used as the mobile phase in a HPLC method to elute LPV and RTV. We found that a solvent ratio of 45 : 5 : 50 (v/v/v) of acetonitrile/methanol/0.02 M TMAP in 0.2% TFA optimized separation of LPV, RTV and EFV. A column temperature of 30 degrees C was necessary for the reproducibility of the analyses. Standard curves were linear in the range 0.060 to 24.06 micro g/ml for LPV, 0.010 to 4.16 micro g/ml for RTV, and 0.047 to 37.44 micro g/ml for EFV. Coefficients of variation (CVs) of LPV, RTV and EFV in intraday and interday assays ranged from 1.5 to 4.0%, 2.5 to 16.8% and 1.0 to 7.7%, respectively. Accuracies ranged from 100 to 110%, 101 to 116% and 99 to 106% for LPV, RTV and EFV, respectively. The extraction recoveries were 77-87, 77-83 and 81-91% for LPV, RTV and EFV, respectively.


Subject(s)
Anti-HIV Agents/analysis , Drug Monitoring/methods , Oxazines/analysis , Pyrimidinones/analysis , Ritonavir/analysis , Alkynes , Anti-HIV Agents/blood , Benzoxazines , Chromatography, High Pressure Liquid , Cyclopropanes , Humans , Lopinavir , Oxazines/blood , Pyrimidinones/blood , Reference Standards , Reproducibility of Results , Ritonavir/blood , Sensitivity and Specificity
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