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

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

Performances of the 6 kits evaluated in this study for a panel of 9 viruses or groups of viruses with reference to the combination of duplex PCR tests (Argene/bioMérieux) used as gold standard.

a<p>Kit having no probe for the detection of HKU1 coronavirus.</p>b<p>Kit having a probe for detecting 229E coronavirus only.</p>c<p>All the tested kits did not detect all the coronavirus types (see notes ^a and ^b).</p

Presentation of the 7 kits used in the study for detecting viruses (see text for correspondence between letters and commercial denominations).

<p>Yes: group of pathogens detected; w diff: with differentiation between types. No: group of pathogens not detected.</p

List of pathogens that were considered to be present in the panel of the 88 specimens according the combination of duplex PCR tests (Argene/bioMérieux) used as gold standard.

a<p>type 1 (n = 2), type 3 (n = 3) and type 4 (n = 5).</p>b<p>type NL63 (n = 6), type OC43 (n = 8), type 229E (n = 1), type HKU1 (n = 6) and untyped (n = 4).</p

Practicability of each kit using the NUCLISENS easyMAG as extraction instrument (see text for correspondence between letters and commercial denominations).

<p>Practicability of each kit using the NUCLISENS easyMAG as extraction instrument (see text for correspondence between letters and commercial denominations).</p

Schematic synopsis of the design of the study.

<p>(See text for correspondence between letters and commercial denominations).</p

Discriminatory ability of soluble factors to classify PCs as belonging to either the adverse effect or control group.

<p>All AUCs with a p-value of <0.0001 (z-test) were considered different from 0.5.</p