[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed CCR5 utilization with retention of the ability to use CXCR4. We also found that the stable association of gp120 with the trimeric envelope glycoprotein complex in R5X4 viruses, as in X4 viruses, is less sensitive to V3 loop changes than gp120-trimer association in R5 viruses.
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 beta2, beta19, beta20, and beta21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.
Journal of Virology 04/2010; 84(7):3147-61. DOI:10.1128/JVI.02587-09 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trimerization of the human immunodeficiency virus (HIV-1) envelope glycoproteins is mediated by the ectodomain of the gp41 transmembrane glycoprotein. Here we investigate oligomer-specific conformations of gp41 by using monoclonal antibodies (MAbs) from HIV-1-infected humans. Human MAbs directed against the cluster I region of gp41 recognized trimeric, dimeric, and monomeric forms of soluble envelope glycoproteins; thus, the integrity of the cluster I epitopes is minimally affected by the oligomeric state. In contrast, human MAbs to the cluster II region were all oligomers specific. One cluster II MAb, 126-6, recognized exclusively the trimeric form of envelope glycoproteins, whereas the others recognized both trimeric and dimeric forms. Thus, a distinct trimer-specific conformation exists in the cluster II region of gp41. Analysis of soluble envelope glycoprotein mutants revealed that gp41 sequences immediately N-terminal to isoleucine 646 contribute to the formation of both the trimer and the trimer-specific conformational epitope.
AIDS research and human retroviruses 04/2009; 25(3):319-28. DOI:10.1089/aid.2008.0213 · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In owl monkeys, the typical retroviral restriction factor of primates, TRIM5alpha, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 and Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection.
[Show abstract][Hide abstract] ABSTRACT: The tripartite motif (TRIM) protein, TRIM5alpha, restricts some retroviruses, including human immunodeficiency virus (HIV-1), from infecting the cells of particular species. TRIM proteins contain RING, B-box, coiled-coil and, in some cases, B30.2(SPRY) domains. We investigated the properties of human TRIM family members closely related to TRIM5. These TRIM proteins, like TRIM5alpha, assembled into homotrimers and co-localized in the cytoplasm with TRIM5alpha. TRIM5alpha turned over more rapidly than related TRIM proteins. TRIM5alpha, TRIM34 and TRIM6 associated with HIV-1 capsid-nucleocapsid complexes assembled in vitro; the TRIM5alpha and TRIM34 interactions with these complexes were dependent on their B30.2(SPRY) domains. Only TRIM5alpha potently restricted infection by the retroviruses studied; overexpression of TRIM34 resulted in modest inhibition of simian immunodeficiency virus (SIV(mac)) infection. In contrast to the other TRIM genes examined, TRIM5 exhibited evidence of positive selection. The unique features of TRIM5alpha among its TRIM relatives underscore its special status as an antiviral factor.
[Show abstract][Hide abstract] ABSTRACT: The coiled-coil domain of the tripartite motif (TRIM) family protein TRIM5alpha is required for trimerization and function as an antiretroviral restriction factor. Unlike the coiled-coil regions of other related TRIM proteins, the coiled coil of TRIM5alpha is not sufficient for multimerization. The linker region between the coiled-coil and B30.2 domains is necessary for efficient TRIM5alpha trimerization. Most of the hydrophilic residues predicted to be located on the surface-exposed face of the coiled coil can be altered without compromising TRIM5alpha antiviral activity against human immunodeficiency virus (HIV-1). However, changes that disrupt TRIM5alpha trimerization proportionately affect the ability of TRIM5alpha to bind HIV-1 capsid complexes. Therefore, TRIM5alpha trimerization makes a major contribution to its avidity for the retroviral capsid, and to the ability to restrict virus infection.
[Show abstract][Hide abstract] ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) gp120 exterior and gp41 transmembrane envelope glycoproteins assemble into trimers on the virus surface that represent potential targets for antibodies. Potent neutralizing antibodies bind the monomeric gp120 glycoprotein with small changes in entropy, whereas unusually large decreases in entropy accompany gp120 binding by soluble CD4 and less potent neutralizing antibodies. The high degree of conformational flexibility in the free gp120 molecule implied by these observations has been suggested to contribute to masking the trimer from antibodies that recognize the gp120 receptor-binding regions. Here we use cross-linking and recognition by antibodies to investigate the conformational states of gp120 monomers and soluble and cell surface forms of the trimeric HIV-1 envelope glycoproteins. The fraction of monomeric and trimeric envelope glycoproteins able to be recognized after fixation was inversely related to the entropic changes associated with ligand binding. In addition, fixation apparently limited the access of antibodies to the V3 loop and gp41-interactive surface of gp120 only in the context of trimeric envelope glycoproteins. The results support a model in which the unliganded monomeric and trimeric HIV-1 envelope glycoproteins sample several different conformations. Depletion of particular fixed conformations by antibodies allowed characterization of the relationships among the conformational states. Potent neutralizing antibodies recognize the greatest number of conformations and therefore can bind the virion envelope glycoproteins more rapidly and completely than weakly neutralizing antibodies. Thus, the conformational flexibility of the HIV-1 envelope glycoproteins creates thermodynamic and kinetic barriers to neutralization by antibodies directed against the receptor-binding regions of gp120.
Journal of Virology 08/2006; 80(14):6725-37. DOI:10.1128/JVI.00118-06 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tripartite motif 5α (TRIM5α) restricts some retroviruses, including human immunodeficiency virus type 1 (HIV-1), from infecting
the cells of particular species. TRIM5α is a member of the TRIM family of proteins, which contain RING, B-box, coiled-coil
(CC), and, in some cases, B30.2(SPRY) domains. Here we investigated the abilities of domains from TRIM proteins (TRIM6, TRIM34,
and TRIM21) that do not restrict HIV-1 infection to substitute for the domains of rhesus monkey TRIM5α (TRIM5αrh). The RING, B-box 2, and CC domains of the paralogous TRIM6 and TRIM34 proteins functionally replaced the corresponding TRIM5αrh domains, allowing HIV-1 restriction. By contrast, similar chimeras containing the components of TRIM21, a slightly more distant
relative of TRIM5, did not restrict HIV-1 infection. The TRIM21 B-box 2 domain and its flanking linker regions contributed
to the functional defectiveness of these chimeras. All of the chimeric proteins formed trimers. All of the chimeras that restricted
HIV-1 infection bound the assembled HIV-1 capsid complexes. These results indicate that heterologous RING, B-box 2, and CC
domains from related TRIM proteins can functionally substitute for TRIM5αrh domains.
Journal of Virology 08/2006; 80(13):6198-206. DOI:10.1128/JVI.00283-06 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primate tripartite motif 5alpha (TRIM5alpha) proteins mediate innate intracellular resistance to retroviruses. In humans, TRIM5 is located in a paralogous cluster that includes TRIM6, TRIM34, and TRIM22. Although TRIM6 and TRIM34 orthologs are found in other mammals, TRIM5 has to date been identified only in primates. Cow cells exhibit early blocks to infection by several retroviruses. We identify a cytoplasmic TRIM protein encoded by LOC505265 that is responsible for the restriction of infection by several lentiviruses and N-tropic murine leukemia virus in cow cells. Susceptibility of N-tropic murine leukemia virus to 505265-mediated restriction is determined primarily by residue 110 of the viral capsid protein. Phylogenetically, cow LOC505265 segregates with the TRIM5/TRIM6/TRIM34 group, but is not an ortholog of known TRIM genes. The B30.2/SPRY domain of 505265 exhibits long variable regions, a characteristic of the proteins encoded by this paralogous group, and shows evidence of positive selection. Apparently, cows have independently evolved a retroviral restriction factor from the same TRIM family that spawned TRIM5 in primates. Particular features of this subset of cytoplasmic TRIM proteins may be conducive to the convergent evolution of virus-restricting factors.
Proceedings of the National Academy of Sciences 06/2006; 103(19):7454-9. DOI:10.1073/pnas.0600771103 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.
[Show abstract][Hide abstract] ABSTRACT: Soluble forms of the trimeric human immunodeficiency virus (HIV-1) envelope glycoproteins are important tools for structural studies and in the construction of improved immunogens. We found that a substantial fraction of soluble envelope glycoprotein trimers contain inter-subunit disulfide bonds (inter-S-S bonds) that render the trimers resistant to heat and denaturing agents. These inter-S-S bonds can be reduced without disrupting the trimers by treatment with a low concentration of beta-mercaptoethanol or DTT. Antibody mapping studies suggest that the soluble HIV-1 envelope glycoprotein trimers lacking the inter-S-S bonds exhibit a conformation closer to that of the native HIV-1 envelope glycoprotein complex. However, reducing these inter-S-S bonds had only modest effects on the inefficient elicitation of neutralizing antibodies by the soluble trimers. These studies provide guidance in improving the resemblance of tractable, soluble forms of the HIV-1 envelope glycoproteins to the native virion spikes.
[Show abstract][Hide abstract] ABSTRACT: The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.
Journal of Virology 06/2004; 78(10):5448-57. DOI:10.1128/JVI.78.10.5448-5457.2004 · 4.44 Impact Factor