Visualization of X4- and R5-Tropic HIV-1 Viruses Expressing Fluorescent Proteins in Human Endometrial Cells: Application to Tropism Study

<div><p>Worldwide most HIV infections occur through heterosexual transmission, involving complex interactions of cell-free and cell-associated particles with cells of the female genital tract mucosa. The ability of HIV-1 to “infect” epithelial cells remains poorly understood. To address this question, replicative-competent chimeric constructs expressing fluorescent proteins and harboring the envelope of X4- or R5-tropic HIV-1 strains were used to “infect” endometrial HEC1-A cells. The virus-cell interactions were visualized using confocal microscopy (CM) at various times post infection. Combined with quantification of viral RNA and total HIV DNA in infected cells, the CM pictures suggest that epithelial cells do not support a complete viral replication cycle: X4-tropic viruses are imported into the nucleus in a non-productive way, whereas R5-tropic viruses transit through the cytoplasm without replication and are preferentially transmitted to susceptible activated peripheral blood mononuclear cells. Within the limit of experiments conducted <i>in vitro</i> on a continued cell line, these results indicate that the epithelial mucosa may participate to the selection of HIV-1 strains at the mucosal level.</p></div

Transmission experiments of HIV from infected HEC-1A cells to uninfected PBMCs by using semi-permeable inserts with 0.3 μm pores.

<p>Confluent HEC1-A cells cultured on the upper part of the inserts were infected for 24h with pBrNL4.3-HXB2-dsRedExpress designed as HxB2 dsRed X4 (A), pBrNL4.3-BaL-eGFP designed as BaL GFP R5 (B) or both chimeric viruses (C). After three washing steps, inserts were then deposited into 24-well plates containing activated PBMCs (15x10⁶ cells per well) and incubated for 24h. PBMCs were then recovered, washed and fixed. An anti-CD26 antibody, labeled with Alexa Fluor™ (AF) 488 in A, AF 555 in B and AF 647 in C, was used for staining PBMCs.</p

Visualization of kinetics of HEC-1A infection by X4 and R5 chimeric viruses by confocal microscopy.

<p>HEC-1A cells were infected for 3, 5, 8, 15 and 24 h by R5-tropic pBrNL4.3-Bal-eGFP chimeric viruses (columns A and B) or X4-tropic pBrNL4.3-HXB2-eGFP chimeric viruses (columns C and D). ZO-1 cellular proteins are colored in red and eGFP signals in green. A and D columns correspond to Differential Interference Contrast (DIC) representations. B and C columns correspond to the green fluorescence channel (green foci correspond to the location of viral particles). Each experiment was done in triplicate, of which a representative example is shown. Scale bars correspond to 10 μm.</p

C3 region sequences of the sequential viruses.

<p>The sequences shown represent the consensus sequence from 3–11 clones. The sequences were derived from the same aliquot of virus culture supernatant that was used in the neutralization assay.</p

CD4 profile of HIV-1 infected study subjects.

<p>All the study subjects were asymptomatic. CD4 counts were determined by FACScan.</p

Chimeric viruses used in this study.

<p>A. Strategy developed for building the chimeric viruses by inserting a PCR-amplified env gene from HXB2 or BaL HIV-1 laboratory strains to Δenv pBrNL4.3 IRES-eGFP/dsRedExpress constructs (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169453#pone.0169453.ref036" target="_blank">36</a>]). B. Quantification of p24 in supernatants of HEK-293 cells after transfection with chimeric viruses: pBrNL4.3-HXB2-eGFP in blue, pBrNL4.3-HXB2-drRedExpress in red, and pBrNL4.3-BaL-eGFP in green. Results are representative of 3 independent transfection experiments and each p24 measurement was performed in duplicate.</p

Neutralization profile of HJ patient plasma and HJ16 mAb in the 24 h/1 h/14d^a extended incubation PBMC assay.

a<p>In this assay virus and plasma or mAb are incubated during 24 hours, followed by an absorption time of 1 hour during which the mAb/virus mixture is co-incubated with donor PBMC. After washing the PBMC cultures are maintained for 14 days.</p>b<p>% Neutralization obtained with 1∶20 plasma dilution or 50 µg/ml mAb during the incubation phase.</p>c<p>≥80% reduction in virus titer is highlighted, E = enhancement of infection.</p

Neutralization curves of anti-V2, anti-CD4bd, and anti-carbohydrate monoclonal antibodies against sequential viruses.

<p>Neutralization sensitivity of HIV-1 subtype B and CRF02_AG viruses obtained from patient ITM60 (a and b), NYU104 (c and d), 3506 (e and f), ITM27 (g and h), and ITM39 (i and j) with anti-V2, anti-CD4bd, and anti-carbohydrate monoclonal antibodies. The dash horizontal line represents 50% neutralization.</p

Neutralization curves of anti-V3 monoclonal antibodies against sequential viruses.

<p>Neutralization sensitivity of HIV-1 subtype B and CRF02_AG viruses obtained sequentially from patient ITM60 (a and b), NYU104 (c and d), 3506 (e and f), ITM27 (g and h), and ITM39 (i and j) with anti-V3 monoclonal antibodies. The dash horizontal line represents 50% neutralization.</p

V2 region sequences of the sequential viruses.

<p>The sequences shown represent the consensus sequence from 3–11 clones. The sequences were derived from the same aliquot of virus culture supernatant that was used in the neutralization assay.</p