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The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope.

ABSTRACT: Current gene transfer protocols for resting CD4(+) T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4(+) T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4(+) T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4(+) T cells. To investigate this, we inoculated resting CD4(+) T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4(+) T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4(+) T cells and suggest that fusion may be the critical step restricting transduction of resting CD4(+) T cells by lentiviral gene therapy vectors.

SUBMITTER: Agosto LM

PROVIDER: S-EPMC2715791 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope.

Agosto Luis M LM Yu Jianqing J JJ Liszewski Megan K MK Baytop Clifford C Korokhov Nikolay N Humeau Laurent M LM O'Doherty Una U

Journal of virology 20090603 16

Current gene transfer protocols for resting CD4(+) T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4(+) T cells without activation. We recently demonst ...[more]

PMID: 19493998

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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.

Project description:HIV-1 R5 variants exploit CCR5 as a coreceptor to infect both T cells and macrophages. R5 viruses that are transmitted or derived from immune tissue and peripheral blood are mainly inefficient at mediating infection of macrophages. In contrast, highly macrophage-tropic (mac-tropic) R5 viruses predominate in brain tissue and can be detected in cerebrospinal fluid but are infrequent in immune tissue or blood even in late disease. These mac-tropic R5 variants carry envelope glycoproteins (Envs) adapted to exploit low levels of CD4 on macrophages to induce infection. However, it is unclear whether this adaptation is conferred by an increased affinity of the Env trimer for CD4 or is mediated by postbinding structural rearrangements in the trimer that enhance the exposure of the coreceptor binding site and facilitate events leading to fusion and virus entry. In this study, we investigated CD4 binding to mac-tropic and non-mac-tropic Env trimers and showed that CD4-IgG binds efficiently to mac-tropic R5 Env trimers, while binding to non-mac-tropic trimers was undetectable. Our data indicated that the CD4 binding site (CD4bs) is highly occluded on Env trimers of non-mac-tropic R5 viruses. Such viruses may therefore infect T cells via viral synapses where Env and CD4 become highly concentrated. This environment will enable high-avidity interactions that overcome extremely low Env-CD4 affinities.IMPORTANCE HIV R5 variants bind to CD4 and CCR5 receptors on T cells and macrophages to initiate infection. Transmitted HIV variants infect T cells but not macrophages, and these viral strains persist in immune tissue even in late disease. Here we show that the binding site for CD4 present on HIV's envelope protein is occluded on viruses replicating in immune tissue. This occlusion likely prevents antibody binding to this site and neutralization of the virus, but it makes it difficult for virus-CD4 interactions to occur. Such viruses probably pass from T cell to T cell via cell contacts where CD4 is highly concentrated and allows infection via inefficient envelope-CD4 binding. Our data are highly relevant for vaccines that aim to induce antibodies targeting the CD4 binding site on the envelope protein.

Dataset Information

The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope.

Publications

The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope.

Similar Datasets

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