Measuring HIV fusion mediated by envelopes from primary viral isolates - PubMed (original) (raw)

Measuring HIV fusion mediated by envelopes from primary viral isolates

Marielle Cavrois et al. Methods. 2011 Jan.

Abstract

Over the course of infection, the human immunodeficiency virus type 1 (HIV-1) continuously adapts in part to evade the host's neutralizing antibody response. Antibodies often target the HIV envelope proteins that mediate HIV fusion to its cellular targets. HIV virions pseudotyped with primary envelopes have often been used to explore the fusogenic properties of these envelopes. Unfortunately, these pseudotyped virions fuse with greatly reduced efficiency to primary cells. Here, we describe a relatively simple strategy to clone primary envelopes into a provirus and increase the sensitivity of the virion-based fusion assay.

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Figures

Figure 1. Comparison of fusion mediated by primary envelopes expressed in trans or in cis

Virions containing BlaM-Vpr were produced by pseudotyping ΔEnv NL4-3 with an expression vector encoding the primary envelopes (pCR3.1-55FPB28a and pCR3.1-109FPB4) or by transfection of a proviral DNA encoding the same primary envelopes (TN6-55FPB28a and TN6-109FPB4). 2 × 106 PBLs or 5 × 105 MDDCs were infected with equal quantities of virions (equivalent of 400 ng of p24Gag) for 2 h at 37°C. (A) FACS plot representing the gating strategy for analysis of fusion of TN6-109FPB4 in CD4 T-cells (first row) or of MDDCs (second row). The BlaM+ cell gate was established based on uninfected samples. Percentages represent the fraction of cells displaying increased blue fluorescence indicative of virion fusion. (B) Histogram representing the level of fusion for experiment performed in triplicate with these envelopes. As a control, cells were treated with TAK-779 for 1 h before the addition of virus. Note the larger increase in fusion obtained when the envelopes were expressed in cis in the proviral construct (TN6-GFP) compared to pseudotyping virions (_Env) in trans. Data presented are representative of results obtained with two independent donors.

Figure 2. Measurement of fusion mediated by envelopes from subtype A, B, C and D

PBLs were infected for 2 h with HIV virions containing BlaM–Vpr in the presence or absence of TAK-779 (500 nM) or AMD3100 (500 nM). The inhibitors were added 1 h prior to infection and maintained throughout the experiment. For the PBLs, HIV-1 fusion was analyzed in CD3+CD4+ subset of cells. The histogram indicates the number of BlaM+ cells. Note the broad range of fusion observed despite the similar amount of virus used to infect the cells. Data are representative of results obtained with two independent donors.

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Measuring HIV fusion mediated by envelopes from primary viral isolates - PubMed (original) (raw)