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Characterization of a dual-tropic human immunodeficiency virus (HIV-1) strain derived from the prototypical X4 isolate HXBc2.

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.

SUBMITTER: Xiang SH

PROVIDER: S-EPMC3594413 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Characterization of a dual-tropic human immunodeficiency virus (HIV-1) strain derived from the prototypical X4 isolate HXBc2.

Xiang Shi-hua SH Pacheco Beatriz B Bowder Dane D Yuan Wen W Sodroski Joseph J

Virology 20130129 1

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 ...[more]

PMID: 23369572

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Project description:The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162DeltaV2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIV(SF162P4) virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (R(h)) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected M(w) of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162DeltaV2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162DeltaV2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162DeltaV2 Env immunogen in a successful HIV vaccine.

Project description:BackgroundHIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.ResultsSoluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.Conclusions3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

Project description:UnlabelledCD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for adaptive antiviral immune responses. Although CD4-independent variants of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have been described that can utilize the coreceptor CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and still can interact with CD4. We describe the derivation of a novel CD4-independent variant of pathogenic SIVmac239, termed iMac239, that was used to derive an infectious R5-tropic SIV lacking a CD4 binding site. Of the seven mutations that differentiate iMac239 from wild-type SIVmac239, a single change (D178G) in the V1/V2 region was sufficient to confer CD4 independence in cell-cell fusion assays, although other mutations were required for replication competence. Like other CD4-independent viruses, iMac239 was highly neutralization sensitive, although mutations were identified that could confer CD4-independent infection without increasing its neutralization sensitivity. Strikingly, iMac239 retained the ability to replicate in cell lines and primary cells even when its CD4 binding site had been ablated by deletion of a highly conserved aspartic acid at position 385, which, for HIV-1, plays a critical role in CD4 binding. iMac239, with and without the D385 deletion, exhibited an expanded host range in primary rhesus peripheral blood mononuclear cells that included CCR5(+) CD8(+) T cells. As the first non-CD4-tropic SIV, iMac239-ΔD385 will afford the opportunity to directly assess the in vivo role of CD4 targeting on pathogenesis and host immune responses.ImportanceCD4 tropism is an invariant feature of primate lentiviruses and likely plays a key role in pathogenesis by focusing viral infection onto cells that mediate adaptive immune responses and in protecting virions attached to cells from neutralizing antibodies. Although CD4-independent viruses are well described for HIV and SIV, these viruses characteristically retain their CD4 binding site and can engage CD4 if available. We derived a novel CD4-independent, CCR5-tropic variant of the pathogenic molecular clone SIVmac239, termed iMac239. The genetic determinants of iMac239's CD4 independence provide new insights into mechanisms that underlie this phenotype. This virus remained replication competent even after its CD4 binding site had been ablated by mutagenesis. As the first truly non-CD4-tropic SIV, lacking the capacity to interact with CD4, iMac239 will provide the unique opportunity to evaluate SIV pathogenesis and host immune responses in the absence of the immunomodulatory effects of CD4(+) T cell targeting and infection.

Dataset Information

Characterization of a dual-tropic human immunodeficiency virus (HIV-1) strain derived from the prototypical X4 isolate HXBc2.

Publications

Characterization of a dual-tropic human immunodeficiency virus (HIV-1) strain derived from the prototypical X4 isolate HXBc2.

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