Transcytosis of HIV-1 through Vaginal Epithelial Cells Is Dependent on Trafficking to the Endocytic Recycling Pathway

BackgroundWhile 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 FindingsIn 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/SignificanceAltogether, 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.

背景 尽管学界普遍认为病毒可通过内吞作用（endocytosis）进入上皮细胞，但目前尚未明确感染性病毒颗粒经阴道上皮细胞转运并从质膜释放的具体生物学机制，这一空白引发了持续的学术争议：一是内吞作用是否仅是某些细胞培养体系中的人为假象，二是鳞状阴道上皮细胞是否与HIV-1的传播过程相关。
研究方法与主要结果 本研究探究了HIV-1用于转胞吞作用（transcytosis）穿过阴道上皮细胞的细胞内转运通路。研究发现，循环内体（recycling endosome）抑制剂可抑制内体小管形成，从而在细胞培养及Transwell小室体系中阻断HIV-1的转胞吞作用。此外，本研究证实：尽管热灭活病毒与天然病毒的内吞效率相当，但热灭活病毒在被内吞后仅被转运至溶酶体通路（lysosomal pathway）进行降解；溶酶体蛋白酶特异性抑制剂（lysosomal protease-specific inhibitor）可阻断灭活病毒颗粒的降解过程。免疫荧光分析（immunofluorescence analysis）不仅证实HIV-1可进入细胞，且天然病毒与热灭活病毒和转铁蛋白（transferrin）呈现出截然不同的共定位（colocalization）模式，这为HIV-1通过循环内体通路穿过阴道上皮细胞提供了确凿证据。
结论与意义 综上，本研究明确了HIV-1在上皮细胞中所利用的精准细胞内转运通路，证实HIV-1经阴道上皮细胞的转胞吞作用是一种真实存在的生物学现象；同时揭示了天然HIV-1与热灭活HIV-1之间细胞内转运通路的差异。若对此展开进一步探索，有望为阻断HIV-1经黏膜上皮的转胞吞作用与传播提供极具价值的理论见解。