Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

Abstract

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission.

HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles

“…Our findings show that HIV endocytosis and macropinocytosis lead to sequestration of virions into late endosomes, including MVB and vacuoles. As we showed recently (Yasen et al, 2017), the interaction of activated lymphocytes with epithelia sequestering HIV-1 initiates the spread of virus from epithelial cells into lymphocytes. Thus, intravesicular HIV-1 sequestration may contribute to the molecular pathogenesis of viral spread from mucosal epithelial cells to virus-susceptible immune cells.…”

“…We have shown that HIV-1 sequestration is gradually reduced at later time points (Yasen et al, 2017). This suggests the possible fusion of vesicles containing HIV-1, including MVB and vacuoles, with highly acidic lysosomes, which may inactivate the virus.…”

“…In our recent work, we have shown that HIV-1 may sequester in the vesicular compartments of epithelial cells without release, i.e., virions are trapped in the endosomes, including MVBs and vacuoles (Yasen et al, 2017). To determine if HIV-1 interaction with the epithelial surface is able to initiate the subsequent sequestration of virus in the vesicles, we studied the role of HSPG, GalCer and TIM-1 in HIV-1 sequestration in tonsil epithelial cells.…”

“…We have shown that HIV-1 sequestration mostly occurs in MVB and vacuoles of epithelial cells (Yasen et al, 2017). To identify the role of initial internalization pathways of virus in its subsequent sequestration in MVB and vacuoles, we silenced expression of the critical proteins of MVB—the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)—and of vacuoles—rabankyrin—by siRNA transfection, respectively.…”

“…Recently, we showed that the majority of infectious virions internalized in tonsil, cervical and foreskin epithelial cells do not cross the epithelium but rather are sequestered in their vesicular/endosomal compartments for several days (Yasen et al, 2017). The interaction of activated lymphocytes with epithelial cells containing HIV-1 facilitates the release of virus and its spread from epithelia into lymphocytes.…”