Human immunodeficiency virus type 1 (HIV-1) superinfection of a cell clone converting it from production of defective to infectious HIV-1 is mediated predominantly by CD4 regions other than the major binding site for HIV-1 glycoproteins

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is not eliminated from patients even after years of antiretroviral therapy, apparently due to the presence of latently infected cells. Here we describe the development of a cell-based system of latency that can be used for high-throughput screening aimed at novel drug discovery to eradicate HIV-1 infection.

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Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.2.495-501.2001 ◽

2001 ◽

Vol 45 (2) ◽

pp. 495-501 ◽

Cited By ~ 65

Author(s):

Atsuko Hachiya ◽

Saori Aizawa-Matsuoka ◽

Mari Tanaka ◽

Yukiko Takahashi ◽

Setsuko Ida ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cell Clone ◽

Drug Susceptibility ◽

Phenotypic Resistance ◽

Phenotypic Assay ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.

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Topotecan inhibits human immunodeficiency virus type 1 infection through a topoisomerase-independent mechanism in a cell line with altered topoisomerase I.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.5.977 ◽

1997 ◽

Vol 41 (5) ◽

pp. 977-981 ◽

Cited By ~ 11

Author(s):

J L Zhang ◽

P L Sharma ◽

C J Li ◽

B J Dezube ◽

A B Pardee ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Cell Line ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Topoisomerase I ◽

Immunodeficiency Virus ◽

Latently Infected ◽

A Cell ◽

Hiv 1

Topotecan (TPT), a known inhibitor of topoisomerase I, has previously been shown to inhibit the replication of several viruses. The mechanism of inhibition was proposed to be the inhibition of topoisomerase I. We report that TPT decreased replication of human immunodeficiency virus type 1 (HIV-1) in CPT-K5, a cell line with a topoisomerase I mutation. TPT inhibited production of HIV-1 RNA and p24 in CPT-K5 and wild-type cells equally effectively. The antiviral effects of TPT were observed not only in the topoisomerase-mutated CPT-K5 line but also in peripheral blood mononuclear cells (PBMC) acutely infected with clinical isolates and in OM10.1 cells latently infected with HIV and activated by tumor necrosis factor alpha. Little toxicity from TPT was noted in HIV-1-infected PBMC and in CPT-K5 and OM10.1 cells as measured by cell growth and proliferation assays. These observations suggest that TPT targets factors in virus replication other than cellular topoisomerase I and inhibits cytokine-mediated activation in latently infected cells by means other than cytotoxicity. These results suggest a potential for TPT and for other camptothecins in anti-HIV therapy alone and in combination with other antiretroviral drugs.

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Potent Rescue of Human Immunodeficiency Virus Type 1 Late Domain Mutants by ALIX/AIP1 Depends on Its CHMP4 Binding Site

Journal of Virology ◽

10.1128/jvi.00314-07 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6614-6622 ◽

Cited By ~ 119

Author(s):

Yoshiko Usami ◽

Sergei Popov ◽

Heinrich G. Göttlinger

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Budding ◽

Rich Domain ◽

Cellular Expression ◽

Immunodeficiency Virus ◽

Hiv 1

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.

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The N-Terminal V3 Loop Glycan Modulates the Interaction of Clade A and B Human Immunodeficiency Virus Type 1 Envelopes with CD4 and Chemokine Receptors

Journal of Virology ◽

10.1128/jvi.74.23.11008-11016.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11008-11016 ◽

Cited By ~ 83

Author(s):

Susan E. Malenbaum ◽

David Yang ◽

Lisa Cavacini ◽

Marshall Posner ◽

James Robinson ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Chemokine Receptors ◽

V3 Loop ◽

Clade B ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We investigated the underlying mechanism by which the highly conserved N-terminal V3 loop glycan of gp120 conferred resistance to neutralization of human immunodeficiency virus type 1 (HIV-1). We find that the presence or absence of this V3 glycan on clade A and B viruses accorded various degrees of susceptibility to neutralization by antibodies to the CD4 binding site, CD4-induced epitopes, and chemokine receptors. Our data suggest that this carbohydrate moiety on gp120 blocks access to the binding site for CD4 and modulates the chemokine receptor binding site of phenotypically diverse clade A and clade B isolates. Its presence also contributes to the masking of CD4-induced epitopes on clade B envelopes. These findings reveal a common mechanism by which diverse HIV-1 isolates escape immune recognition. Furthermore, the observation that conserved functional epitopes of HIV-1 are more exposed on V3 glycan-deficient envelope glycoproteins provides a basis for exploring the use of these envelopes as vaccine components.

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Identification and Characterization of a Peptide That Specifically Binds the Human, Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody b12

Journal of Virology ◽

10.1128/jvi.75.14.6692-6699.2001 ◽

2001 ◽

Vol 75 (14) ◽

pp. 6692-6699 ◽

Cited By ~ 67

Author(s):

Michael B. Zwick ◽

Lori L. C. Bonnycastle ◽

Alfredo Menendez ◽

Melita B. Irving ◽

Carlos F. Barbas ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Human Monoclonal Antibody ◽

Recombinant Polypeptide ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.

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Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human Immunodeficiency Virus Type 1 gp120

Journal of Virology ◽

10.1128/jvi.77.10.5863-5876.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5863-5876 ◽

Cited By ~ 78

Author(s):

Michael B. Zwick ◽

Paul W. H. I. Parren ◽

Erica O. Saphire ◽

Sarah Church ◽

Meng Wang ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dimensional Structure ◽

Side Chains ◽

Molecular Features ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT IgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities.

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The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.77.23.12507-12522.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12507-12522 ◽

Cited By ~ 51

Author(s):

Sébastien Violot ◽

Saw See Hong ◽

Dina Rakotobe ◽

Caroline Petit ◽

Bernard Gay ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dependent Manner ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Hiv 1

ABSTRACT Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4+ cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

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A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

Journal of Virology ◽

10.1128/jvi.73.11.8966-8974.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 8966-8974 ◽

Cited By ~ 128

Author(s):

Alexandra Trkola ◽

Jamie Matthews ◽

Cynthia Gordon ◽

Tom Ketas ◽

John P. Moore

Keyword(s):

Human Immunodeficiency Virus ◽

Cell Line ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Neutralization Assay ◽

Immunodeficiency Virus ◽

A Cell ◽

Cxcr4 Coreceptor ◽

Hiv 1

ABSTRACT We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.

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Identification of an APOBEC3G Binding Site in Human Immunodeficiency Virus Type 1 Vif and Inhibitors of Vif-APOBEC3G Binding

Journal of Virology ◽

10.1128/jvi.00204-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 13235-13241 ◽

Cited By ~ 74

Author(s):

Andrew Mehle ◽

Heather Wilson ◽

Chengsheng Zhang ◽

Andrew Jay Brazier ◽

Mark McPike ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Binding Assays ◽

Immunodeficiency Virus ◽

N Terminus ◽

Hiv 1

ABSTRACT The APOBEC3 cytidine deaminases are potent antiviral factors that restrict replication of human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif binds APOBEC3G and APOBEC3F and targets these proteins for ubiquitination by forming an E3 ubiquitin ligase with cullin 5 and elongins B and C. The N-terminal region of Vif is required for APOBEC3G binding, but the binding site(s) is unknown. To identify the APOBEC3G binding site in Vif, we established a scalable binding assay in a format compatible with development of high-throughput screens. In vitro binding assays using recombinant proteins identified Vif peptides and monoclonal antibodies that inhibit Vif-APOBEC3G binding and suggested involvement of Vif residues 33 to 83 in APOBEC3G binding. Cell-based binding assays confirmed these results and demonstrated that residues 40 to 71 in the N terminus of Vif contain a nonlinear binding site for APOBEC3G. Mutation of the highly conserved residues His42/43 but not other charged residues in this region inhibited Vif-APOBEC3G binding, Vif-mediated degradation of APOBEC3G, and viral infectivity. In contrast, mutation of these residues had no significant effect on Vif binding and degradation of APOBEC3F, suggesting a differential requirement for His42/43 in Vif binding to APOBEC3G and APOBEC3F. These results identify a nonlinear APOBEC3 binding site in the N terminus of Vif and demonstrate that peptides or antibodies directed against this region can inhibit Vif-APOBEC3G binding, validating the Vif-APOBEC3 interface as a potential drug target.

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