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

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 in vitro 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.

Persistent But Not Replicating HIV-1 Cell-Associated DNA in Semen of Long-Term ART Experienced Men.

BACKGROUND: The semen of HIV-1 infected men represents the main vector of HIV-1 spread following sexual transmission of cell-free or cell-associated virions. OBJECTIVE: The present study aimed to assess the impact of HAART on HIV-1 RNA/DNA and on inflammatory environment in the semen of long-term HAART-experienced men. METHODS: Forty-five paired samples of semen and blood were obtained from 37 consenting men, 10 untreated and 27 under HAART. Blood and seminal HIV RNA and DNA loads were quantified by the Abbott RealTime m2000rt assay and an inhouse real-time PCR protocol, respectively. Tat/rev/nef intra-cellular mRNA was tested by qualitative PCR. Interleukin (IL)- 1ß, IL-2, IL-6, IL-7, IL-8, IL-10, GM-CSF and TNFα were quantified in 20 paired samples by Bio-plex® assay. RESULTS: No semen was found HIV RNA positive in men under HAART. Twenty-six percent of semen samples from HAART-experienced men remained positive for HIV DNA. Seminal HIV DNA was significantly associated with the duration of infection and the HIV DNA load in blood. No seminal mononuclear cells were found positive for intracellular HIV RNA in HAART experienced men. All the tested chemokines exhibited significantly higher concentration in semen than in blood in both treated and untreated men. No effect of HAART on cytokines/chimiokines loads was observed. CONCLUSION: These results demonstrate the efficacy of HAART on the reduction of seminal RNA HIV-1 loads despite the persistence of local inflammation. Moreover, in our hands the seminal cell-associated virus reservoir was not reactivated in an inflammatory environment was not productive and its reactivation seems unlikely.

Selective transmigration of monocyte-associated HIV-1 across a human cervical monolayer and its modulation by seminal plasma.

OBJECTIVE: To analyse the transmigration of immune cells infected by HIV-1 across the epithelial monolayer using the endometrial human endometrial carcinoma (HEC)-1A cell line and to study the influence of seminal plasma in this process. DESIGN: After sexual intercourse involving a male partner infected by HIV-1, a selection process has been shown to lead to a predominant transmission of the R5 phenotype despite the presence of X4 and R5 strains in semen. Transmigration of HIV-infected monocytes present in semen may represent a pertinent mechanism that could explain this tropism selection. METHODS: Epithelial monolayer crossing was studied by using HEC-1A epithelial cells cultured on permeable support and monocyte-enriched or lymphocyte-enriched populations of cells infected or not by HIV R5 or X4 strains. Transmigrating cells were quantified and analysed for their ability to transmit HIV infection to immune target cells. The effect of HIV-negative seminal plasma on cell transmigration was analysed. RESULTS: A preferential passage of the R5 strain associated with monocyte-enriched populations was observed together with the ability of this strain to transmit infection. Seminal plasma was found able to decrease the epithelial crossing of immune cells by enhancing transepithelial resistance and by increasing the adherence of immune cells to the monolayer. CONCLUSION: The preferential transmigration of HIV R5 strains associated with monocytes across the endocervical monolayer may explain the predominant transmission of the R5 strains after sexual intercourse. By its capacity to modulate the tightness of the epithelial structure, seminal plasma reinforces this selection process.