thelial cells or traverse the epithelial barrier dissimulated in infected cells to establish a primary infection. Thus, the literature remains ambiguous as to whether HIV-1 proviral DNA integrates into the epithelial cell genome and whether productive infection ensues. Notably, a mechanism has been described recently to explain the difference between oral transmission in adults and infants using ex vivo cell models, whereby adult epithelium coats HIV-1 particles with human b-defensins 2 and 3, rendering the cell-free virus less infectious. The same group also established that the paucistratified fetal and infant oral epithelium was more permissive to HIV-1 transcytosis as compared with healthy multistratified adult oral epithelium. Given the ambiguous literature on this fundamentally important topic, we aimed to compare oral and vaginal epithelial cells using identical assay systems to determine whether cell-free HIV-1 binds, enters and integrates into epithelial cells, whether productive infection ensues, and whether sequestered infectious virus can be transferred to permissive cells. We utilized epithelial cell lines representative of two oral sites that are rarely investigated with regard to oral transmission but are one of the first cell types that are likely to come into contact with HIV-1 in the oral cavity and compared them with female genital epithelium. Buccal, pharyngeal and vulvo-vaginal epithelial cells, all from stratified squamous cell origin, were utilized to permit convenient and direct comparisons to be made with regard to HIV-1 life cycle events. We demonstrate that oral and vaginal epithelial cells were able to capture X4 and R5 virus but that integration of the viral genome into epithelial cell DNA and de novo virus production were not detected. However, VSV-G-packaged HIV-1 was replication competent in all epithelial cell lines. Notably, the three epithelial cell types were able to transfer infectious virus to permissive cells either directly or after transcytosis of HIV-1 across the epithelium, which in vivo would permit infection of immune cells in the sub-mucosa and dissemination of HIV-1 in the body. Methods Ethics Statement Primary epithelial cells were obtained from wisdom tooth extractions and were kindly provided by Maxine Partridge and collection approved by the Guy’s Research Ethics Committee. 13679187 Informed written consent was obtained from each participant. Cell lines, primary cells, viruses and virus-like particles Human oral buccal and pharyngeal and vulvovaginal carcinoma cell lines and 293T cells were obtained from the American Type Culture Collection and European Collection of Cell Cultures. A431 epithelial cells have different reported origins but are routinely used to represent the vaginal mucosa. Human glioma cells expressing human CD4 and CXCR4 or CCR5 have been previously described. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl cells, PM-1 cells, 
C8166 T cells, JTLRG-R5 and HIV-1 molecular clones pYU2 and pLAI.2. Peripheral blood mononuclear cells were isolated from apheresis cones by centrifugation over a Ficoll-Paque density gradient. The HIV-1 JNJ-7777120 web gag-pol expression vector p8.91 19615387 and the vesicular stomatitis virus envelope protein expression vector pMDG were kindly provided by Didier Trono. The HIV gp160 envelope vectors, pYU2 and pSVIII 89.6 ) were a gift from Professor Greg Towers, University College London. The retr
