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

p7 ATMI mutant viruses display increased E2 secretion and decreased RNA and core secretion.

<p>Huh7.5 cells were electroporated with RNAs from parental or Jc1 HAHALp7 mutant HCVcc viruses expressed alone or with wild-type p7 or HAHALp7. Analyses were performed at 72h post-electroporation. All data are normalized by percentage of HCV-positive virus producer cells obtained as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.g003" target="_blank">Fig 3A</a>. Results obtained with other p7 ATMI mutant viruses are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.s004" target="_blank">S4 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.s005" target="_blank">S5 Fig</a>. (<b>A</b>) Levels of secreted E2 determined by quantitative western blot following GNA lectin pull down of cell supernatants. (<b>B</b>) Levels of secreted core as determined by CMIA (Chemiluminescent Microparticle ImmunoAssay). (<b>C</b>) Levels of secreted HCV RNAs as determined by RT-qPCR. (<b>D</b>) Ratio of extracellular E2 to intracellular E2. (<b>E</b>) Ratio of extracellular E2 to extracellular core. (<b>F</b>) Ratio of extracellular E2 to extracellular HCV RNA. (<b>G</b>) Specific infectivity relative to E2 amounts. (<b>H</b>) Specific infectivity relative to core amounts. (<b>I</b>) Specific infectivity relative to RNA amounts. All values are displayed relative to infectivity and expression of E2, core or RNA values determined in the supernatants of Jc1 virus-electroporated cells (<b>A-I</b>). Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

Model of p7 role in assembly and envelopment of HCV particles.

<p>(<b>1</b>) HCV assembly takes place at the ER membrane close to HCV replication complexes-containing double membrane vesicles (DMV) and to cytosolic lipid droplets (LD) harboring core and NS5A at their surface. Upon translation of the HCV genome, E1E2p7NS2 complexes form and accumulate at the ER membrane. (<b>2</b>) p7, <i>via</i> its N-terminal extremity, regulates (thin grey arrow) the interaction of NS2 with NS5A. (<b>3</b>) This interaction may allow the release of E1E2 from E1E2p7NS2 complexes at the assembly site of nascent viral particles on the one hand and co-recruitment of core and RNA to E1E2 on the other hand. Subsequently, the E1E2-free p7NS2NS5A complex leaves whereas a new E1E2p7NS2 complex reaches the assembly area (dotted arrows). (<b>4</b>) The process is reiterated with p7 from incoming E1E2p7NS2 complexes regulating the encountering of NS2 and NS5A, which leads to the further release of E1E2 and core/RNA that accumulate at the assembly site. (<b>5</b>) The process is repeated until the formation of a viral particle that buds in the ER lumen. (<b>6</b>) The NS2NS5A complex, regulated by p7, may recruit ESCRT components to induce scission of the nascent particle until full envelopment and egress.</p

HCVcc mutants with modified p7 amino-terminus impair NS2 and NS5A association.

<p>Huh7.5 cells expressing RNAs from parental <i>vs</i>. Jc1 HAHALp7 mutant viruses expressed alone or with wild-type p7 were analyzed at 72h. (<b>A</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV core (red), NS5A (green) and NS2 (blue). (<b>B</b>) Co-localization analysis from (<b>A</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>C</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV NS2 (red), E2 (green) and NS5A (blue). (<b>D</b>) Co-localization analysis from (<b>C</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>E</b>) Cell lysates were incubated with protein A/G agarose beads after pre-treatment, or not (beads), with NS2 antibodies. Immuno-precipitated complexes were eluted and analyzed by quantitative western blot (see representative western blot to the left). The graphs show the levels of NS5A proteins co-immuno-precipitated by NS2 antibodies normalized to the amount of immuno-precipitated NS2 in Jc1 or Jc1 HAHALp7 virus-expressing cells. The values are displayed relative to association of NS5A to NS2 immuno-precipitated from Jc1 virus-expressing cells. Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

HCVcc mutants with modified p7 amino-terminus impair NS2 and NS5A association.

<p>Huh7.5 cells expressing RNAs from parental <i>vs</i>. Jc1 HAHALp7 mutant viruses expressed alone or with wild-type p7 were analyzed at 72h. (<b>A</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV core (red), NS5A (green) and NS2 (blue). (<b>B</b>) Co-localization analysis from (<b>A</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>C</b>) Confocal microscopy of Huh7.5 cells expressing Jc1 or Jc1 HAHALp7 viruses and stained for HCV NS2 (red), E2 (green) and NS5A (blue). (<b>D</b>) Co-localization analysis from (<b>C</b>), displaying the Pearson’s correlation coefficients and the Manders’ overlap coefficients, as indicated. (<b>E</b>) Cell lysates were incubated with protein A/G agarose beads after pre-treatment, or not (beads), with NS2 antibodies. Immuno-precipitated complexes were eluted and analyzed by quantitative western blot (see representative western blot to the left). The graphs show the levels of NS5A proteins co-immuno-precipitated by NS2 antibodies normalized to the amount of immuno-precipitated NS2 in Jc1 or Jc1 HAHALp7 virus-expressing cells. The values are displayed relative to association of NS5A to NS2 immuno-precipitated from Jc1 virus-expressing cells. Data represent mean values ± SEM. The numbers of experiments performed are indicated below the graphs.</p

HCVcc mutants with modified p7 amino-termini prevent infectivity.

<p><b>(A)</b> Schematic representation of JFH1 and Jc1 HCVcc mutants in E2-p7 junction. The first helix of p7 is shown in the light grey box. The insertions of residues are shown in dark grey and the substitutions are underlined. (<b>B-D</b>) Western blot analysis of Huh7.5 cells electroporated with RNAs from HCVcc viruses encoding wild-type or E2p7 cleavage mutants. <b>(B)</b> At 72h post-electroporation, cells were lysed and digested with endoH glycosidase before western blotting using 3/11 antibodies against E2. <b>(C)</b> The relative ratios of E2p7 precursors <i>vs</i>. free E2 species are indicated. <b>(D)</b> The HAHALp7 protein and the E2HAHALp7 (white arrows) and E2HAHALp7NS2 (black arrows) precursors were revealed using an HA tag antibody in lysates of cells electroporated with the indicated HCVcc constructs. <b>(E)</b> The extracellular (black) and intracellular (grey) infectivity levels for all mutants are represented. <b>(F)</b> The extracellular (black) and intracellular (grey) infectivity levels, normalized after determining the proportion of HCV-positive virus producer cells (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006774#ppat.1006774.g003" target="_blank">Fig 3A</a>), of JFH1 HAHALp7 or p7-T2 mutant viruses expressed alone or with wild-type p7 (+ p7) are represented relative to infectivity of parental JFH1 virus. <b>(G)</b> The extracellular (black) and intracellular (grey) infectivity levels, normalized by determining the proportion of HCV-positive virus producer cells, of Jc1 HAHALp7 mutant virus expressed alone or with wild-type p7 or HAHALp7, as indicated, are represented relative to infectivity of parental Jc1 virus. Data are displayed as means ± SEM. The numbers of experiments performed are indicated below the graphs.</p

p7 ATMI mutant viruses do not alter E2, core, NS4B, NS5A and HCV RNA(+) clustering.

<p><b>(A)</b> Confocal microscopy of Huh7.5 cells infected with Jc1 or Jc1 HAHALp7 viruses. At 72h post-infection, cells were fixed and stained for HCV core (red), E2 (green), NS4B (blue, left panels), NS5A (blue, right panels) and nuclei (grey). <b>(B)</b> Quantification of size (left) and number (right) of co-localized core/E2/NS4B or core/E2/NS5A structures. (<b>C</b>) Confocal microscopy of Huh7.5 expressing Jc1 or Jc1 HAHALp7 viruses. At 72h post-infection, cells were fixed and stained for HCV core (green), NS4B (blue, left panels), NS5A (blue, right panels) and nuclei (grey). HCV RNA(+) were stained by FISH (red). <b>(D)</b> Quantification of percentage of core/NS4B or core/NS5A structures co-localizing with RNA(+) (left), of RNA(+) co-localizing with core (middle), or of RNA(+) co-localizing with NS4B/NS5A structures. Scale bars of panels and zooms from squared area represent 10μm and 2μm, respectively. For each condition, over 20 cells were quantified.</p