oviral packaging vector pCSGW encoding green fluorescent protein was a kind gift from Adrian Thrasher, Institute of Child Health, University College London, UK. All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum , 100 U of penicillin per mL, and 100 mg of streptomycin per mL at 37uC and 5% CO2. NP2 cells were additionally supplemented with 1 mg/mL of G418 and 1 mg/ mL of puromycin. Virus preparation Viral vectors used for production of VSV-G and HIV gp160 pseudotyped HIV were prepared by transient transfection of 293T cells using a protocol adapted from Besnier et al.. Briefly, 293T cells were 1260907-17-2 site seeded at 95% confluency in a 10 cm2 dish and the following day cells were transfected with 3 mg each of pMDG and p8.91 and 4.5 mg of pCSGW using the polyanionic Epithelial Cells Binding and Transfer Infectious HIV-1 transfection reagent Jet PEI according to the manufacturer’s instructions. After 24 h the media was replaced and 48, 72 and 96 h post-transfection virion-containing culture supernatants were harvested and filtrated through a 0.45 mm pore size membrane and stored in aliquots at 80uC until required. Production of infectious stocks of live virus was performed by transient transfection of 293T cells as described above, with 5 mg of the infectious molecular clone pLAI.2 and or pYU2 used per transfection. For trypsin sensitivity experiments and detection of integration into epithelium, YU2 virus was grown in NP2-R5 and JLTRG-R5 cells while LAI virus was grown in C8166 cells for 1- 2 weeks with addition of fresh medium until cells showed cytopathic effects and were then harvested and frozen in aliquots. HIV-1 receptor expression by flow cytometry TZM-bl, NP2-X4, NP2-R5, TR146, FaDu and A431 resting cells were washed with PBS and incubated with 0.02% EDTA for 530 min. Detached cells were washed thoroughly with PBS supplemented with 1% BSA and 0.01% azide, and resuspended at 16106 cells in 1 mL wash buffer. To identify surface expressed HIV-1 receptors and co-receptors, 100 mL of cells were incubated at room temperature for 1 h with mouse antihuman CD4, CCR5, CXCR4, DC-SIGN monoclonal antibodies, GalCer or heparan sulfate proteoglycan monoclonal antibodies. Primary antibodies were detected with goat anti-mouse IgG conjugated with fluorescein isothiocyanate . After thorough 13679187 washing, cells were fixed in 200 mL 4% formaldehyde and the percentage of FITC-expressing cells was determined by flow cytometry. Virus titration Infectious virus stock titers were determined by plaque assay. Briefly, TZM-bl cells were cultured overnight and incubated with eight replicates of ten serial dilutions of virus stock in a total of 100 mL growth media per well. After 48 h, virus supernatant was removed and the cells were fixed with 0.05% glutaraldehyde for 5 min at room temperature and washed twice with phosphatebuffered saline. Expression of b-galactosidase was determined by staining cells with X-Gal stain and incubating culture plates at 37uC for 2 h. Virus infectivity was estimated as plaque forming units per mL. Titration of VSV-G pseudotyped HIV-1 and HIV gp160 pseudotyped HIV-1 was carried out using 293T cells or NP2 cells, respectively. Cells were seeded at 1 19615387 x 105 
cells/well and cultured overnight at 37uC. Serial dilutions of virus supernatant were applied to the cells and incubated overnight. The following day the media was exchanged and 48 h after transduction with virion-containing culture supern
