High throughput functional analysis of HIV-1 env genes without cloning

Kirchherr, Jennifer; Lu, Xiaozhi; Kasongo, Webster; Chalwe, Victor; Mwananyanda, Lawrence; Musonda, Rosemary; Xia, Shi-Mao; Scearce, Richard M.; Liao, Hua‐Xin; Montefiori, David C.; Haynes, Barton F.; Gao, Feng

doi:10.1016/j.jviromet.2007.02.015

Cited by 46 publications

(54 citation statements)

References 20 publications

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“…Eighty-three HIV-1-transmitting mothers from the WITS cohort were selected according to the following inclusion criteria: no Maternal autologous virus and mAb isolation. HIV-1 env sequences were generated from plasma of a nontransmitting mother by single genome amplification at 2 months postpartum and produced as pseudoviruses (27). V3-specific B cells were isolated from autologous peripheral blood mononuclear cells (PBMCs) at 6 months postpartum (first available) by flow cytometric sorting using a ConB V3 peptide tetramer.…”

Section: Methodsmentioning

confidence: 99%

Maternal HIV-1 envelope–specific antibody responses and reduced risk of perinatal transmission

Permar¹,

Fong²,

Vandergrift³

et al. 2015

J. Clin. Invest.

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112

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Section: Methodsmentioning

confidence: 99%

Maternal HIV-1 envelope–specific antibody responses and reduced risk of perinatal transmission

Permar¹,

Fong²,

Vandergrift³

et al. 2015

J. Clin. Invest.

Self Cite

112

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“…We used a yeast gap-repair homologous recombination system to generate recombinant HIV-1 that contained the dominant full-length env sequence of plasma virus obtained from subject 07 (Sub07) at weeks 0, 16, 19, and 28 as described previously (12,54). Recombinant virus that incorporated HIV-1 envelopes from subjects 57 (Sub57) and 85 (Sub85) were constructed by a modification of a previously described method (15,20). Briefly, the cytomegalovirus (CMV) promoter was amplified and attached to a 265-bp segment of the rev gene from pNL43 using overlap PCR.…”

Section: Methodsmentioning

confidence: 99%

HIV-1 Clinical Isolates Resistant to CCR5 Antagonists Exhibit Delayed Entry Kinetics That Are Corrected in the Presence of Drug

Putcharoen

Lee

Henrich

et al. 2012

J Virol

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HIV CCR5 antagonists select for env gene mutations that enable virus entry via drug-bound coreceptor. To investigate the mechanisms responsible for viral adaptation to drug-bound coreceptor-mediated entry, we studied viral isolates from three participants who developed CCR5 antagonist resistance during treatment with vicriviroc (VCV), an investigational small-molecule CCR5 antagonist. VCV-sensitive and -resistant viruses were isolated from one HIV subtype C-and two subtype B-infected participants; VCV-resistant isolates had mutations in the V3 loop of gp120 and were cross-resistant to TAK-779, an investigational antagonist, and maraviroc (MVC). All three resistant isolates contained a 306P mutation but had variable mutations elsewhere in the V3 stem. We used a virus-cell ␤-lactamase (BlaM) fusion assay to determine the entry kinetics of recombinant viruses that incorporated full-length VCV-sensitive and -resistant envelopes. VCV-resistant isolates exhibited delayed entry rates in the absence of drug, relative to pretherapy VCV-sensitive isolates. The addition of drug corrected these delays. These findings were generalizable across target cell types with a range of CD4 and CCR5 surface densities and were observed when either populationderived or clonal envelopes were used to construct recombinant viruses. V3 loop mutations alone were sufficient to restore virus entry in the presence of drug, and the accumulation of V3 mutations during VCV therapy led to progressively higher rates of viral entry. We propose that the restoration of pre-CCR5 antagonist therapy HIV entry kinetics drives the selection of V3 loop mutations and may represent a common mechanism that underlies the emergence of CCR5 antagonist resistance. Human immunodeficiency virus entry is a competition at the target cell membrane between viral inactivation and successful binding to CD4 and a coreceptor, either CCR5 or CXCR4 (16,30,32,33,37,38). Engagement of receptor and coreceptor by gp120 and the subsequent rearrangement of gp41, the HIV fusion glycopeptides, leads to fusion (18,21,29,57,60,63). Smallmolecule antagonists that bind CCR5 and inhibit gp120-CCR5 binding through an allosteric mechanism have been developed (9,23,51,53,56,61). Maraviroc (MVC) is an FDA-approved CCR5 antagonist, and vicriviroc (VCV) is an investigational compound whose development has been halted (10, 47).HIV can escape CCR5 antagonism through the outgrowth of preexisting CXCR4-using populations or by the selection of resistance mutations (2, 52, 58). Mutations in the third hypervariable loop (V3 loop) of HIV-1 gp120 most commonly cause CCR5 antagonist resistance, but no canonical sites or amino acid substitutions have been identified; resistance mutations from one isolate generally do not confer resistance when transferred into unrelated env backbones (12, 17, 24, 26, 27, 34-36, 49, 54). This varied nature of HIV CCR5 antagonist genotypic resistance contrasts with resistance patterns established in other antiretroviral drug classes, for which canonical mutations (M184V in r...

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“…A custom-made mixture of two HLA-C-specific siRNAs (Matucci et al, 2008) has been shown previously to be effective in silencing the expression of HLA-C in the TZMbl cell line (Kirchherr et al, 2007). RNA analysis (Fig.…”

Section: Hla-c Sirna Silencing In Cell Linesmentioning

confidence: 99%

HLA-C is necessary for optimal human immunodeficiency virus type 1 infection of human peripheral blood CD4 lymphocytes

Baroni

Matucci

Scarlatti

et al. 2009

Journal of General Virology

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The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P50.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the 11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses. INTRODUCTIONThree recent whole-genome association studies have demonstrated and confirmed that minor allele variants of singlenucleotide polymorphisms in the major histocompatibility complex (MHC; in particular HLA-C) gene regions are associated with a lower viral load at the set point and additionally with delayed human immunodeficiency virus type 1 (HIV-1) disease progression (Fellay et al., 2007;Limou et al., 2009;van Manen et al., 2009). These findings, which echo a previous report on the association between HLA-C alleles and rapid progression to AIDS (Carrington et al., 1999), have prompted wide speculations on the role of HLA (and HLA-C in particular) in HIV biology and immunology. HLA-C differs from HLA-A and -B because HIV-1 selectively downregulates HLA-A and -B, but does not significantly affect HLA-C (or HLA-E). Therefore, infected cells display reduced detection by both cytotoxic lymphocytes and natural killer (NK) cells (Cohen et al., 1999). The hypothesis that HLA-C might exert an effect directly on HIV infectivity was originally suggested by De Santis et al. (1996), exploiting the peculiar specificity of monoclonal antibody (mAb) L31 to show that normal levels of Env-driven fusion were restored in HLA-C transfectants (Grassi et al., 1991;Setini et al., 1996) of an MHC-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs), the natural target of HIV-1. Two different kinds o...

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High throughput functional analysis of HIV-1 env genes without cloning

Cited by 46 publications

References 20 publications

Maternal HIV-1 envelope–specific antibody responses and reduced risk of perinatal transmission

Maternal HIV-1 envelope–specific antibody responses and reduced risk of perinatal transmission

HIV-1 Clinical Isolates Resistant to CCR5 Antagonists Exhibit Delayed Entry Kinetics That Are Corrected in the Presence of Drug

HLA-C is necessary for optimal human immunodeficiency virus type 1 infection of human peripheral blood CD4 lymphocytes

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