scholarly journals Variants of Human Immunodeficiency Virus Type 1 That Efficiently Use CCR5 Lacking the Tyrosine-Sulfated Amino Terminus Have Adaptive Mutations in gp120, Including Loss of a Functional N-Glycan

2005 ◽  
Vol 79 (7) ◽  
pp. 4357-4368 ◽  
Author(s):  
Emily J. Platt ◽  
Danielle M. Shea ◽  
Patrick P. Rose ◽  
David Kabat
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Strong Interactions ◽  
Amino Terminus ◽  
Adaptive Mutations ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT By selecting the R5 human immunodeficiency virus type 1 (HIV-1) strain JR-CSF for efficient use of a CCR5 coreceptor with a badly damaged amino terminus [i.e., CCR5(Y14N)], we previously isolated variants that weakly utilize CCR5(Δ18), a low-affinity mutant lacking the normal tyrosine sulfate-containing amino-terminal region of the coreceptor. These previously isolated HIV-1JR-CSF variants contained adaptive mutations situated exclusively in the V3 loop of their gp120 envelope glycoproteins. We now have weaned the virus from all dependency on the CCR5 amino terminus by performing additional selections with HeLa-CD4 cells that express only a low concentration of CCR5(Δ18). The adapted variants had additional mutations in their V3 loops, as well as one in the V2 stem (S193N) and four alternative mutations in the V4 loop that eliminated the same N-linked oligosaccharide from position N403. Assays using pseudotyped viruses suggested that these new gp120 mutations all made strong contributions to use of CCR5(Δ18) by accelerating a rate-limiting CCR5-dependent conformational change in gp41 rather than by increasing viral affinity for this damaged coreceptor. Consistent with this interpretation, loss of the V4 N-glycan at position N403 also enhanced HIV-1 use of a different low-affinity CCR5 coreceptor with a mutation in extracellular loop 2 (ECL2) [i.e., CCR5(G163R)], whereas the double mutant CCR5(Δ18,G163R) was inactive. We conclude that loss of the N-glycan at position N403 helps to convert the HIV-1 envelope into a hair-trigger form that no longer requires strong interactions with both the CCR5 amino terminus and ECL2 but efficiently uses either site alone. These results demonstrate a novel functional role for a gp120 N-linked oligosaccharide and a high degree of adaptability in coreceptor usage by HIV-1.

scholarly journals Adaptive Mutations in the V3 Loop of gp120 Enhance Fusogenicity of Human Immunodeficiency Virus Type 1 and Enable Use of a CCR5 Coreceptor That Lacks the Amino-Terminal Sulfated Region

2001 ◽  
Vol 75 (24) ◽  
pp. 12266-12278 ◽  
Author(s):  
Emily J. Platt ◽  
Shawn E. Kuhmann ◽  
Patrick P. Rose ◽  
David Kabat
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Terminus ◽  
V3 Loop ◽  
Wild Type ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To identify sites in gp120 that interact with the CCR5 coreceptor and to analyze the mechanisms of infection, we selected variants of the CCR5-dependent JRCSF molecular clone of human immunodeficiency virus type 1 (HIV-1) that adapted to replicate in HeLa-CD4 cells that express the mutant coreceptor CCR5(Y14N) or CCR5(G163R), which were previously shown to bind purified gp120-CD4 complexes only weakly. Correspondingly, these mutant CCR5s mediate infections of wild-type virus only at relatively high cell surface concentrations, demonstrating a concentration-dependent assembly requirement for infection. The plots of viral infectivity versus concentration of coreceptors had sigmoidal shapes, implying involvement of multiple coreceptors, with an estimated stoichiometry of four to six CCR5s in the active complexes. All of the adapted viruses had mutations in the V3 loops of their gp120s. The titers of recombinant HIV-1 virions with these V3 mutations were determined in previously described panels of HeLa-CD4 cell clones that express discrete amounts of CCR5(Y14N) or CCR5(G163R). The V3 loop mutations did not alter viral utilization of wild-type CCR5, but they specifically enhanced utilization of the mutant CCR5s by two distinct mechanisms. Several mutant envelope glycoproteins were highly fusogenic in syncytium assays, and these all increased the efficiency of infection of the CCR5(Y14N) or CCR5(G163R) clonal panels without enhancing virus adsorption onto the cells or viral affinity for the coreceptor. In contrast, V3 loop mutation N300Y was selected during virus replication in cells that contained only a trace of CCR5(Y14N) and this mutation increased the apparent affinity of the virus for this coreceptor, as indicated by a shift in the sigmoid-shaped infectivity curve toward lower concentrations. Surprisingly, N300Y increased viral affinity for the second extracellular loop of CCR5(Y14N) rather than for the mutated amino terminus. Indeed, the resulting virus was able to use a mutant CCR5 that lacks 16 amino acids at its amino terminus, a region previously considered essential for CCR5 coreceptor function. Our results demonstrate that the role of CCR5 in infection involves at least two steps that can be strongly and differentially altered by mutations in either CCR5 or the V3 loop of gp120: a concentration-dependent binding step that assembles a critical multivalent virus-coreceptor complex and a postassembly step that likely involves a structural rearrangement of the complex. The postassembly step can severely limit HIV-1 infections and is not an automatic consequence of virus-coreceptor binding, as was previously assumed. These results have important implications for our understanding of the mechanism of HIV-1 infection and the factors that may select for fusogenic gp120 variants during AIDS progression.

scholarly journals Amino-Terminal Substitutions in the CCR5 Coreceptor Impair gp120 Binding and Human Immunodeficiency Virus Type 1 Entry

1998 ◽  
Vol 72 (1) ◽  
pp. 279-285 ◽  
Author(s):  
Tatjana Dragic ◽  
Alexandra Trkola ◽  
Steven W. Lin ◽  
Kirsten A. Nagashima ◽  
Francis Kajumo ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Surface Glycoprotein ◽  
Alanine Scanning Mutagenesis ◽  
Cc Chemokine ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The CC-chemokine receptor CCR5 is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of CD4+ cells and interacts directly with the viral surface glycoprotein gp120. Although receptor chimera studies have provided useful information, the domains of CCR5 that function for HIV-1 entry, including the site of gp120 interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of CCR5 to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish CCR5-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair Env-mediated membrane fusion and the gp120-CCR5 interaction. Of these three residues, only D11 is necessary for CC-chemokine-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites on CCR5 are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal CCR5 domain.

scholarly journals Structural Requirements for Recognition of the Human Immunodeficiency Virus Type 1 Core during Host Restriction in Owl Monkey Cells

2005 ◽  
Vol 79 (2) ◽  
pp. 869-875 ◽  
Author(s):  
Brett M. Forshey ◽  
Jiong Shi ◽  
Christopher Aiken
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cells ◽  
Host Restriction ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Owl Monkey ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of simian cells is restricted at an early postentry step by host factors whose mechanism of action is unclear. These factors target the viral capsid protein (CA) and attenuate reverse transcription, suggesting that they bind to the HIV-1 core and interfere with its uncoating. To identify the relevant binding determinants in the capsid, we tested the capacity of viruses containing Gag cleavage site mutations and amino acid substitutions in CA to inhibit restriction of a wild type HIV-1 reporter virus in owl monkey cells. The results demonstrated that a stable, polymeric capsid and a correctly folded amino-terminal CA subunit interface are essential for saturation of host restriction in target cells by HIV-1 cores. We conclude that the owl monkey cellular restriction machinery recognizes a polymeric array of CA molecules, most likely via direct engagement of the HIV-1 capsid in target cells prior to uncoating.

scholarly journals Differential CD4/CCR5 Utilization, gp120 Conformation, and Neutralization Sensitivity between Envelopes from a Microglia-Adapted Human Immunodeficiency Virus Type 1 and Its Parental Isolate

2001 ◽  
Vol 75 (8) ◽  
pp. 3568-3580 ◽  
Author(s):  
Julio Martı́n ◽  
Celia C. LaBranche ◽  
Francisco González-Scarano
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Syncytium Formation ◽  
Luciferase Reporter ◽  
Recombinant Viruses ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infects and induces syncytium formation in microglial cells from the central nervous system (CNS). A primary isolate (HIV-1BORI) was sequentially passaged in cultured microglia, and the isolate recovered (HIV-1BORI-15) showed high levels of fusion and replicated more efficiently in microglia (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. González-Scarano, J. Virol. 70:7654–7662, 1996). The parent and adapted viruses used CCR5 as coreceptor. Recombinant viruses demonstrated that the syncytium-inducing phenotype was associated with four amino acid differences in the V1/V2 region of the viral gp120 (J. T. C. Shieh, J. Martin, G. Baltuch, M. H. Malim, and F. González-Scarano, J. Virol. 74:693–701, 2000). We produced luciferase-reporter, env-pseudotyped viruses using plasmids containing env sequences from HIV-1BORI, HIV-1BORI-15, and the V1/V2 region of HIV-1BORI-15 in the context of HIV-1BORI env (named rBORI, rB15, and rV1V2, respectively). The pseudotypes were used to infect cells expressing various amounts of CD4 and CCR5 on the surface. In contrast to the parent recombinant, the rB15 and rV1V2 pseudotypes retained their infectability in cells expressing low levels of CD4 independent of the levels of CCR5, and they infected cells expressing CD4 with a chimeric coreceptor containing the third extracellular loop of CCR2b in the context of CCR5 or a CCR5 Δ4 amino-terminal deletion mutant. The VH-rB15 and VH-rV1V2 recombinant viruses were more sensitive to neutralization by a panel of HIV-positive sera than was VH-rBORI. Interestingly, the CD4-induced 17b epitope on gp120 was more accessible in the rB15 and rV1V2 pseudotypes than in rBORI, even before CD4 binding, and concomitantly, the rB15 and rV1V2 pseudotypes were more sensitive to neutralization with the human 17b monoclonal antibody. Adaptation to growth in microglia—cells that have reduced expression of CD4 in comparison with other cell types—appears to be associated with changes in gp120 that modify its ability to utilize CD4 and CCR5. Changes in the availability of the 17b epitope indicate that these affect conformation. These results imply that the process of adaptation to certain tissue types such as the CNS directly affects the interaction of HIV-1 envelope glycoproteins with cell surface components and with humoral immune responses.

scholarly journals A Patch of Positively Charged Amino Acids Surrounding the Human Immunodeficiency Virus Type 1 Vif SLVx4Yx9Y Motif Influences Its Interaction with APOBEC3G

2009 ◽  
Vol 83 (17) ◽  
pp. 8674-8682 ◽  
Author(s):  
Gongying Chen ◽  
Zhiwen He ◽  
Tao Wang ◽  
Rongzhen Xu ◽  
Xiao-Fang Yu
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Terminal ◽  
Critical Residue ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Positively Charged

ABSTRACT The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K22x3K26x3Y30x9YRHHY44) and a cluster of hydrophobic residues (V55xIPLx4-5LxΦx2YWxL72) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.

scholarly journals CCR5 and CXCR4 Usage by Non-Clade B Human Immunodeficiency Virus Type 1 Primary Isolates

2002 ◽  
Vol 76 (6) ◽  
pp. 3059-3064 ◽  
Author(s):  
Daniah A. D. Thompson ◽  
Emmanuel G. Cormier ◽  
Tatjana Dragic
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Fusion ◽  
Amino Terminal ◽  
Cxcr4 Usage ◽  
Clade B ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT CCR5 and CXCR4 usage has been studied extensively with a variety of clade B human immunodeficiency virus type 1 (HIV-1) isolates. The determinants of CCR5 coreceptor function are remarkably consistent, with a region critical for fusion and entry located in the CCR5 amino-terminal domain (Nt). In particular, negatively charged amino acids and sulfated tyrosines in the Nt are essential for gp120 binding to CCR5. The same types of residues are important for CXCR4-mediated viral fusion and entry, but they are dispersed throughout the extracellular domains of CXCR4, and their usage is isolate dependent. Here, we report on the determinants of CCR5 and CXCR4 coreceptor function for a panel of non-clade B isolates that are responsible for the majority of new HIV-1 infections worldwide. Consistent with clade B isolates, CXCR4 usage remains isolate dependent and is determined by the overall content of negatively charged and tyrosine residues. Residues in the Nt of CCR5 that are important for fusion and entry of clade B isolates are also important for the entry of all non-clade B HIV-1 isolates that we tested. Surprisingly, we found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects fusion and entry of all non-clade B isolates tested. This points to a different mechanism of CCR5 usage by these viruses and may have important implications for the development of HIV-1 inhibitors that target CCR5 coreceptor function.

scholarly journals Determinants of Human Immunodeficiency Virus Type 1 Resistance to gp41-Derived Inhibitory Peptides

1998 ◽  
Vol 72 (2) ◽  
pp. 986-993 ◽  
Author(s):  
Laurence T. Rimsky ◽  
Diane C. Shugars ◽  
Thomas J. Matthews
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mechanism Of Action ◽  
Synthetic Peptide ◽  
Heptad Repeat ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051–1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1IIIB and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain.

scholarly journals Cooperation of the V1/V2 and V3 Domains of Human Immunodeficiency Virus Type 1 gp120 for Interaction with the CXCR4 Receptor

2001 ◽  
Vol 75 (12) ◽  
pp. 5457-5464 ◽  
Author(s):  
Béatrice Labrosse ◽  
Carole Treboute ◽  
Anne Brelot ◽  
Marc Alizon
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Direct Interaction ◽  
V3 Loop ◽  
Terminal Part ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry is triggered by the interaction of the gp120 envelope glycoprotein with a cellular chemokine receptor, either CCR5 or CXCR4. We have identified different mutations in human CXCR4 that prevent efficient infection by one HIV-1 strain (NDK) but not another (LAI) and sought to define these strain-dependent effects at the gp120 level. The lack of activity toward the NDK strain of the HHRH chimeric CXCR4 in which the second extracellular loop (ECL2) derived from the rat CXCR4 and of CXCR4 with mutations at an aspartic acid in ECL2 (D193A and D193R) was apparently due to the sequence of the third variable loop (V3) of gp120, more precisely, to its C-terminal part. Indeed, substitution of the LAI V3 loop or only its C-terminal part in the NDK gp 120 context was sufficient to restore usage of the HHRH, D193A, and D193R receptors. The same result was achieved upon mutation of a single lysine residue of the NDK V3 loop to alanine (K319A) but not to arginine (K319R). These results provide a strong case for a direct interaction between the gp120 V3 loop and the ECL2 domain of CXCR4. By contrast, V3 substitutions had no effect on the inability of NDK to infect cells via a mutant CXCR4 in which the amino-terminal extracellular domain (NT) is deleted. In experiments with a set of chimeric NDK-LAI gp120s, the V1/V2 region from LAI gp120 was both necessary and sufficient for usage of the NT-deleted CXCR4. Different variable domains of gp120 can therefore cooperate for a functional interaction with CXCR4.

scholarly journals Alanine Substitutions of Polar and Nonpolar Residues in the Amino-Terminal Domain of CCR5 Differently Impair Entry of Macrophage- and Dualtropic Isolates of Human Immunodeficiency Virus Type 1

1998 ◽  
Vol 72 (4) ◽  
pp. 3464-3468 ◽  
Author(s):  
Gwénaël E. E. Rabut ◽  
Jason A. Konner ◽  
Francis Kajumo ◽  
John P. Moore ◽  
Tatjana Dragic
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Gp120 Binding ◽  
Immunodeficiency Virus ◽  
Amino Terminal Domain ◽  
Hiv 1

ABSTRACT Multiple extracellular domains of the CC-chemokine receptor CCR5 are important for its function as a human immunodeficiency virus type 1 (HIV-1) coreceptor. We have recently demonstrated by alanine scanning mutagenesis that the negatively charged residues in the CCR5 amino-terminal domain are essential for gp120 binding and coreceptor function. We have now extended our analysis of this domain to include most polar and nonpolar amino acids. Replacement of alanine with all four tyrosine residues and with serine-17 and cysteine-20 decrease or abolish gp120 binding and CCR5 coreceptor activity. Tyrosine-15 is essential for viral entry irrespective of the test isolate. Substitutions at some of the other positions impair the entry of dualtropic HIV-1 isolates more than that of macrophagetropic ones.

scholarly journals Reciprocal Modulatory Interaction between Human Immunodeficiency Virus Type 1 Tat and Transcription Factor NFAT1

1999 ◽  
Vol 19 (5) ◽  
pp. 3645-3653 ◽  
Author(s):  
Fernando Macián ◽  
Anjana Rao
Keyword(s):  
Human Immunodeficiency Virus ◽  
Transcription Factor ◽  
Host Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Protein ◽  
Amino Terminal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by interactions between both viral and host factors. These interactions are also responsible for changes in the expression of many host cell genes, including cytokines and other immune regulators, which may account for the state of immunological dysregulation that characterizes HIV-1 infection. We have investigated the role of a host cell protein, the transcription factor NFAT1, in HIV-1 pathogenesis. We show that NFAT1 interacts with Tat and that this interaction, which involves the major transactivation domain of NFAT1 and the amino-terminal region of Tat, results in a reciprocal modulatory interplay between the proteins: whereas Tat enhances NFAT1-driven transcription in Jurkat T cells, NFAT1 represses Tat-mediated transactivation of the HIV-1 long terminal repeat (LTR). Moreover, NFAT1 binds to the κB sites on the viral LTR and negatively regulates NF-κB-mediated activation of HIV-1 transcription, by competing with NF-κB1 for its binding sites on the HIV-1 LTR. Tat-mediated enhancement of NFAT1 transactivation may explain the upregulation of interleukin 2 and other cytokines that occurs during HIV-1 infection. We discuss the potentially opposing roles of NFAT1 and another family member, NFAT2, in regulating gene transcription of HIV-1 and endogenous cytokine genes.

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