Transcytosis of HIV-1 through vaginal epithelial cells is dependent on trafficking to the endocytic recycling pathway

Abstract

Background: While it is accepted that viruses can enter epithelial cells by endocytosis, the lack of an established biological mechanism for the trafficking of infectious virions through vaginal epithelial cells and their release from the plasma membrane has contributed to ongoing controversy about whether endocytosis is a mere artifact of some cell culture systems and whether squamous vaginal epithelial cells are even relevant as it pertains to HIV-1 transmission.

Methodology/principal findings: In this study, we investigated the intracellular trafficking pathway that HIV-1 exploits to transcytose vaginal epithelial cells. The reduction of endosome tubulation by recycling endosome inhibitors blocked transcytosis of HIV-1 in a cell culture and transwell system. In addition, we demonstrate that although heat-inactivated virus was endocytosed as efficiently as native virus, heat-inactivated virus was trafficked exclusively to the lysosomal pathway for degradation following endocytosis. Lysosomal protease-specific inhibitors blocked the degradation of inactivated virions. Immunofluorescence analysis not only demonstrated that HIV-1 was inside the cells but the different colocalization pattern of native vs. heat inactivated virus with transferrin provided conclusive evidence that HIV-1 uses the recycling pathway to get across vaginal epithelial cells.

Conclusions/significance: Altogether, our findings demonstrate the precise intracellular trafficking pathway utilized by HIV-1 in epithelial cells, confirms that HIV-1 transcytosis through vaginal epithelial cells is a biological phenomenon and brings to light the differential intracellular trafficking of native vs heat-inactivated HIV-1 which with further exploration could prove to provide valuable insights that could be used in the prevention of transcytosis/transmission of HIV-1 across the mucosal epithelia.

Figure 1. Native and heat-inactivated HIV-1 enters vaginal epithelial cells in a dose- and time-dependent manner.

VK2 cells were incubated at 37°C, 5% CO₂ with indicated amounts of native or heat-inactivated HIV-1, and then the cells were thoroughly washed with PBS and incubated with 0.05% trypsin for 3 min at room temperature to ensure removal of non-internalized virus. (A) HIV-1 IIIB uptake in VK2 cells after exposure to increasing amounts of virus for 4 h was analyzed by Western blot using a p24 antibody with actin staining as the loading control. (B) Time course of viral uptake in vaginal epithelial cells after exposure to 100 ng HIV-1 IIIB. (C) Viral uptake of equal amounts (100 ng) of HIV-1 IIIB obtained from H9 cells or HIV-1 IIIB, YU2 and NL4-3 obtained from Jurkat CCR5 cells. Each experiment in this and subsequent figures was performed at least three times, and representative blots are presented.

Figure 2. Inactivation of HIV-1 does not affect viral uptake but inhibits viral release from vaginal epithelial cells.

VK2 cells were incubated at 37°C, 5% CO₂ with 100 ng native or heat-inactivated HIV-1 IIIB for 4 h. Cells were then thoroughly washed with PBS and incubated with 0.05% trypsin for 3 min at room temperature to ensure removal of non-internalized virus. Fresh media was added to individual wells, and virus-containing supernatants and cells were harvested at the indicated time points. Viral uptake in cells was analyzed by Western blot (A, B). Virus release in culture supernatants was analyzed by qRT-PCR (C). MAGI assay as used to determine viral infectivity (F) of virus found in supernatant from Day 1. Values are means ± SEM of three independent experiments.

Figure 3. Native HIV-1 utilizes the tubulation-dependent endocytic recycling pathway.

VK2 cells were incubated at 37°C, 5% CO₂ with 100 ng native or heat-inactivated HIV-1 IIIB for 4 h and then thoroughly washed and incubated with 0.05% trypsin to remove non-internalized virus. Fresh media was added to cells with increasing amounts (0, 1, 2.5, 5, 10 uM) of BEL. Cells and supernatants were collected 24 h later for analysis. Intracellular viral protein content was analyzed by Western blot (A, B). Viral release was measured by qRT-PCR (C). VK2 cells were transfected with plasmids expressing DN Rab11-GFP or pCDNA 3.1-GFP as control. Cells were then inoculated with 100 ng HIV-1 IIIB 48 h post-transfection. After washing away the non-internalized inoculum, fresh media was then added. On day 2, levels of intracellular virus and released virus in the supernatant were analyzed using Western blotting (D) and qRT-PCR (E), respectively, as described in Materials and methods. Values are means ± SEM of three independent experiments.

Figure 4. Both native and heat-inactivated virus enter the lysosomal degradation pathway.

VK2 cells were pretreated with a cocktail of lysosomal inhibitors (final concentration: 29 µM pepstatin A, 52 µM leupeptin and 69 µM E-64) for 32 h at 37°C, 5% CO₂. Cells were then inoculated with native or heat-inactivated HIV-1 for 4 h at 37°C, 5% CO₂ and then washed and trypsinized to remove non-internalized virus. Inhibitors were maintained in culture during the viral exposure. After virus removal, lysosomal inhibitors were added back to the fresh media, and cells and supernatants were collected at indicated time points for analysis by Western blot (A, B) and qRT-PCR (C), respectively. Values are means ± SEM of three independent experiments.

Figure 5. HIV-1 entry into vaginal epithelial cells is envelope-independent, and inhibition of heat-inactivated HIV-1 release is envelope-independent.

VK2 cells were incubated for 4-inactivated or untreated NL4-3 or NL4-3 ΔEnv virus. Cells were then thoroughly washed with PBS and incubated with 0.05% trypsin for 3 min at room temperature to ensure removal of non-internalized virus. Fresh media was then added to individual wells, and the supernatants were harvested at indicated time points. Virus release was determined by qRT-PCR (A, C). Intracellular viral protein was analyzed by Western blot (B). Relative virus levels in supernatants were determined using qRT-PCR. Values are means ± SEM of three independent experiments.

Figure 6. Vaginal epithelial cells support HIV-1 Transcytosis.

VK2 cells were grown on a transwell insert containing 3.0 µm pores. Heat-inactivated or untreated NL4-3 ΔENV (100 ng) was then added to the apical chamber and the viral levels in media of the basal chamber were assayed after 1 h incubation at 37°C (A). Native or Heat inactivated HIV-1 IIIB (B) or PBMC derived HIV-1 Ba-L (C) were added to the apical chamber and viral levels in media of the basal chamber were assayed after 1 h. NL4-3 ΔENV (D)HIV-1 IIIB (E) or PBMC derived HIV-1 Ba-L (F) were added to the apical chamber and incubated for 1 h. Media from the apical and basal chambers were removed and replaced with fresh media containing 1 µM BEL. Viral levels in media of the basal chamber were assayed after 24 h using qRT-PCR. Values are means ± SEM of three or more independent experiments

Figure 7. HIV-1 colocalizes with the recycling endocytic marker transferrin.

VK2 cells were plated on glass coverslips in a 12-well plate and grown 48 h at 37°C, 5% CO₂. Native or heat-inactivated HIV-1 IIIB was then added to the cells and incubated for 4 h then subjected to confocal microscopy as described in Materials and methods after removal of non-internalized virus. HIV-1 is shown in green and transferrin is shown in red. Bar graph represents the percentage cells positive for colocalization from the random counting of 162 cells exposed to native and 170 cells exposed to heat-inactivated virus. Arrows points to colocalization signals. Inset is a magnification of the signal that is inside the box.