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Early Infection HIV-1 Envelope V1-V2 Genotypes Do Not Enhance Binding or Replication in Cells Expressing High Levels of α4β7 Integrin

Etemad, Behzad PhD*; Gonzalez, Oscar A. BS*; McDonough, Sean BS; Pena-Cruz, Victor BS*; Sagar, Manish MD*

JAIDS Journal of Acquired Immune Deficiency Syndromes: November 1st, 2013 - Volume 64 - Issue 3 - p 249–253
doi: 10.1097/QAI.0b013e3182a06ddd
Brief Report: Basic and Translational Science

Abstract: It has been postulated that HIV-1 envelope properties, such as shorter and less-glycosylated V1-V2 loops commonly observed among non–subtype B early-transmitted viruses, promote utilization of the gut homing integrin α4β7. This property potentially confers an advantage to some HIV-1 variants early after acquisition. We found that replication-competent recombinant viruses incorporating HIV-1 subtype A compact and less-glycosylated early versus chronic phase V1-V2 loops demonstrated no significant difference in binding to α4β7 high CD8+ T cells or replication in α4β7 high CD4+ T cells. Integrin α4β7 usage does not select for shorter less-glycosylated envelopes during transmission.

*Department of Medicine, Division of Infectious Disease, Boston University School of Medicine, Boston, MA; and

Dana Farber Cancer Research Center, Boston, MA.

Correspondence to: Manish Sagar, MD, Boston University School of Medicine, Evans Biomedical Research Center, 650 Albany Street, EBRC-647, Boston, MA 02118-2518 (e-mail:

Supported by National Institute of Health Grant AI1077473 (M.S.).

The authors have no conflicts of interest to disclose.

Received October 19, 2012

Accepted June 13, 2013

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High-level replication in gut-associated lymphoid tissue likely plays an important role in establishing a systemic infection early after HIV-1 acquisition.1,2 Infection of CD4+ T cells expressing the gut homing integrin α4β7 potentially facilitates HIV-1 migration from mucosal sites to gut-associated lymphoid tissue.3,4 Enhanced integrin α4β7 reactivity has been linked to specific envelope characteristics, such as smaller V1-V2 loops and transmission-associated predicted N-linked glycosylation sites (PNGS).5 These envelope genotypes are commonly observed in subtypes A, C, and D but not in subtype B early-transmitted viruses.6–12 Enrichment of viruses with these envelope signatures suggests that specific viruses are preferentially favored for acquisition, and α4β7 integrin utilization potentially confers fitness for transmission.13

Studies suggesting that compact and less-glycosylated envelope V1-V2 loops enhance α4β7 utilization have been primarily conducted with HIV-1 envelope surface unit monomer, gp120, and not native envelope trimers on virus particles.5 One recent study showed that replication of a small number (n = 6) of subtype C transmitted/founder (T/F) and unrelated chronic infection (n = 4) strains were not inhibited by blocking the α4β7 integrin, suggesting that the infecting viruses do not use the α4β7 integrin more efficiently.14 Because the T/F and chronic isolates were obtained from different subjects, they did not examine α4β7 utilization differences among closely related viruses with and without the transmission-associated genotypes, such as compact and less-glycosylated V1-V2 loops. In this study, we directly assessed the influence of transmission-associated envelope V1-V2 loop signatures on α4β7 utilization.

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Subjects and Viruses

Demographics of the heterosexually infected subjects with subtype A HIV-1 and the envelope sequences examined in this study have been detailed previously.6,15 We evaluated the most commonly amplified V1-V2 loop from both time points and another atypical chronic sequence in 2 subjects (QA203 and QB424). The V1-V2 loops were placed in a Q23-17 subtype A HIV-1 envelope backbone as previously described.6,15 The chimeric envelopes were incorporated into a plasmid containing Q23-17 HIV sequences from the primer binding site (PBS) to the 3’ long terminal repeat (LTR), pCMV-Q23-17-PBS→LTR, using yeast gap-repair homologous recombination as previously described.16–19 Replication-competent viruses were generated by cotransfecting HEK293T cells with a plasmid containing the subject V1-V2 envelope within pCMV-Q23-17-PBS→LTR and another plasmid with Q23-17 sequences from 5′ LTR to early portion of gag, pCMV-Q23-17-LTR→Gag4.16 The 293T transfection supernatants were passaged on activated CD4+ T cells for a maximum of 7 days to generate high-titer peripheral blood mononuclear cell-derived viruses. Virus stocks were titered on TZM-bl cells as previously described.6,20

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Binding and Replication Assessment

Primary CD4+ and CD8+ T cells were isolated from HIV-1 negative blood donor’s buffy coats using antibody-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) according to manufacturer’s instructions. Both CD8+ and CD4+ T cells were cultured with 2% phytohemagglutinin, 20 μg/mL recombinant human IL-2 with or without 10 nM retinoic acid (RA) for 6 days. Approximately 1 × 105 infectious particles were incubated independently with 1 × 106 CD8+ T cells and 1 × 106 CD4+ T cells at 4°C for 1 hour in binding buffer [10 mM HEPES, 150 mM NaCl (HBS Buffer) buffer with100 μM CaCl2 and 1 mM MnCl2]. In some cases, cells were preincubated with the specified antibodies at 37°C for 30 minutes before virus exposure. The CD4+ and CD8+ T cells were washed with RPMI 3 times to remove unbound virus. RNA was isolated from the CD8+ T-cell incubations using the QIAAMP Viral RNA kit (QIAGEN, Germantown, MD). HIV-1 copies were quantified using quantitative reverse transcription-polymerase chain reaction using previously described methods.21,22 The CD4+ T-cell cultures were incubated at 37°C 5% CO2. Supernatants were collected 3 days postinfection and not at later times to probe early replication differences, which were more likely to be affected by α4β7 usage. Supernatant infectious particles concentrations were estimated by infecting 1 × 104 TZM-bl cells with 8 serial 2-fold dilutions, and cells were examined for beta-galactosidase production using Galacto-Light Plus System (Applied Biosystems, Grand Island, NY) after 2 days. A linear interpolated curve of the relative light units versus supernatant dilution was used to estimate relative light units per microliter.

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Statistical Analysis

Values in the presence of RA and no antibody were compared with other cell culture conditions using the Wilcoxon rank sum test matched per donor cells. Early and chronic infection median values were compared among all subjects using the matched pairs Wilcoxon rank sum test. In 2 subjects (QA203 and QA424) where 2 chronic infection V1-V2 loops were examined a median of the 2 chronic infection values was used in the comparisons. All analysis was done with GraphPad Prism 5. All P values were based on a 2-sided test.

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The α4β7 cell surface mean fluorescence intensity increased in the presence when compared with the absence of RA in cells from 13 of the 17 blood donors’ buffy coats (Figs. 1A, B). We only used donor cells with FACS confirmed RA-induced increase in α4β7 expression for further experiments. Although cells expressed α4β7 under both conditions, α4β7 mean fluorescence intensity was significantly higher in the presence when compared with the absence of RA in CD4+ (mean fold change, 2.5; range, 1.3–4.5) and CD8+ T cells (mean fold change, 2.1; range, 1.5–3.2) (P = 0.0002 for both comparisons). We examined α4β7 binding in CD8+ as opposed to in CD4+ T cells with preblocked CD4 receptor because both cell types expressed relatively similar levels of α4β7. In addition, this strategy eliminated the possibility of low-level virus binding to the unblocked CD4 receptor.



We first examined Bal envelope virus and HIV-1SF162, which have been shown to have α4β7 integrin reactivity.3,4 Binding and replication in cells incubated with different antibodies specific for α4β7 (2B4 and Act 1) and a control antibody specific for β1 (P4G11) or in the absence of RA was compared with CD8+ and CD4+ T cells grown in the presence of RA and no antibody (set as 100%). Bal and SF162 RNA copies recovered from virus exposed to CD8+ T cells was significantly lower in the presence of 2B4 (P = 0.03), Act 1 (P = 0.03), and in the absence of RA (P = 0.03) but was not significantly different in the presence of P4G11 (P > 0.05) (Fig. 1C). SF162 replication in CD4+ T cells was also significantly lower in the presence of Act 1 (P = 0.008), 2B4 (P = 0.008), and in the absence of RA (P = 0.02) but was not significantly different with P4G11 (P = 0.6) (Fig. 1D). Bal replication was significantly lower when CD4+ T cells were preexposed to Act 1 (P = 0.03) but not significantly different in the presence of 2B4 (P = 0.06), absence of RA (P = 0.4), or with P4G11 (P = 1.0) (Fig. 1D). Thus, virus binds to CD8+ T cells potentially because of baseline α4β7 expression, and this attachment increases with RA-induced higher gut homing receptor levels. Similar to published reports, replication was not reduced among all virus isolates in the presence of α4β7-blocking antibodies or lower gut homing receptor levels.14,23

A previous study showed that a HIV-1 subtype A envelope gp120 monomer incorporating V1-V2 loops from early in infection [QA203M1, also previously referred to as QA203D(M1)] showed around 20-fold greater binding to the α4β7 integrin compared with a gp120 with V1-V2 segments from the chronic phase of disease [QA203M41 also previously referred to as QA203B(M41)].5,6 We compared binding and replication between the QA203D(M1) and QA203B(M41) V1-V2 loop envelope viruses as opposed to gp120 monomers. Because the producer cells determine the types of glycosylation present on a virus envelope, we derived all viruses with subject-specific envelope V1-V2 loops from physiologically relevant peripheral blood mononuclear cell cultures.5 QA203D(M1) and QA203B(M41) V1-V2 loops encode 61 and 73 amino acids with 5 and 8 PNGS, respectively (Fig. 2A). Similar to the gp120 data, we found that QA203B(M41) virus attachment was lower compared with QA203D(M1) virus (set at 100%) (P = 0.06, n = 5 replicates) (Fig. 2B).5 The QA203B(M41) virus demonstrated significantly lower replication in CD4+ T cells expressing high levels of α4β7 compared with QA203D(M1) (set as 100%) (P = 0.008, n = 8 replicates) (Fig. 2C).



To assess the generalizability of this finding, we examined the influence of other longitudinally isolated V1-V2 loops on α4β7 utilization (Fig. 2A). All envelopes possessed the tripeptide V2 motif associated with α4β7 binding.3 The chronic when compared with the early infection V1-V2 loops contained significantly more PNGS (P = 0.03) and were longer (P = 0.09) although length differences did not reach statistical significance. In only 2 of the 8 subjects (QA203 and QB670), higher amounts of RNA was recovered from the viruses with early relative to the chronic infection V1-V2 (Fig. 2B). In aggregate, early when compared to chronic infection viruses showed no significant differences in binding to α4β7 high CD8+ T cells (P = 0.2). In all subjects, except QC449 and QC890, viruses with early relative to chronic infection V1-V2 replicated more efficiently in α4β7 high CD4+ T cells (Fig. 2C). These chronic versus early differences were significant (P < 0.05) for some isolates [QA203A(M41), QA203B(M41), QB424D(M31), QB596M(M24), and QB670B(M46)]. In aggregate, however, there was no significant replication difference among the viruses with longitudinally collected V1-V2 (P = 0.3). Because majority of early compared with chronic infection viruses demonstrated increased replication in α4β7 high CD4+ T cells, we examined if they were more sensitive to α4β7 blocker, Act 1. Early versus chronic virus V1V2 replication was not inhibited to a greater extent in the presence of Act 1 (P = 0.15). Thus, Act 1 did not inhibit replication among most isolates, and a number of V1-V2 chimeric viruses actually demonstrated enhanced replication, similar to a previous published report.14 Infectious virus in α4β7 high CD4+ T-cell culture day 3 supernatant did not have significant correlation with number of RNA recovered from α4β7 high CD8+ T cells (ρ = 0.04, P = 0.8, Spearman rank correlation).

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In this study, we examined the influence of longitudinally collected envelope V1-V2 loops on binding to α4β7 high CD8+ T cells and replication in α4β7 high CD4+ T cells. The majority of early infection V1-V2 loops were smaller with a significantly lower number of PNGS compared with the chronic phase variants. The early when compared with chronic V1-V2 loop viruses did not consistently demonstrate higher binding or replication among the cells with high levels of the gut homing receptor. In aggregate, our results suggest that attachment to the integrin or replication in α4β7 expressing CD4+ T cells does not influence the observed enrichment of subtype A viruses with compact and less-glycosylated V1-V2 loop envelopes early after HIV-1 acquisition.

Reactivity to the α4β7 integrin has been primarily examined among different gp120s and not infectious viruses. Functional trimers on replication competent virus are likely structurally different than envelope surface unit monomers. Interestingly, both a chimeric gp120 and recombinant functional envelope on infectious virus with an early when compared with a chronic QA203 V1-V2 demonstrated greater binding to α4β7-expressing cells.5 This suggests that α4β7 binding may not be immensely different between gp120s and envelope trimers although this needs to be confirmed in more isolates. One recent study has examined α4β7 utilization among subtype C T/F and unrelated chronic phase viruses.14 In contrast to this study, we assessed intrasubject subtype A envelope V1-V2 loop influence on α4β7 usage. Combined results from both studies suggest that gut homing receptor utilization does not favor the selection of variants with specific envelope genotypes, such as compact and less-glycosylated V1-V2 loops commonly observed during non–subtype B HIV-1 transmission.6–12

The biological mechanism for the observed selection of specific viruses remains unclear. Because infection efficiency in α4β7 high CD4+ T cells is likely not a transmission phenotype, prevention strategies aimed at blocking HIV-1 envelope–α4β7 interaction may not stop acquisition. Furthermore, recently described V2-directed antibodies shown as a correlate of protection in an HIV-1 vaccine trial likely do not prevent infection by blocking α4β7 binding.24–26

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The authors thank all the subjects who have contributed samples for these studies and the NIH AIDS Research and Reference Reagent Program for TZM-bl cells and Act 1 antibody.

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gut homing integrin α4β7; mucosal transmission; HIV-1 envelope; selection at transmission; gut-associated lymphoid tissue

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