Restricted HIV-1 Infection in TERT-2 Oral Keratinocytes

Oral keratinocytes protect against HIV-1 infection during oral-sexual contacts and breast-feeding. Yet, keratinocytes transfer HIV-1 to peripheral blood mononuclear cells (PBMCs) in vitro. It is unclear, however, whether HIV-1 replicates in oral keratinocytes. Objective: To test the hypothesis that HIV entry and replication are restricted in oral keratinocytes (OKF6/TERT-2; TERT-2). Methods: To determine receptivity to HIV-1, TERT-2 cells were analyzed for putative HIV receptors/co-receptors. TERT-2 cells were engineered to express CD4 to rescue a potential receptor restriction. Potential innate immune restrictions were analyzed by qRT-PCR. HIV-1 IIIb and BaL integration and specific transcripts were detected by nested PCR and qRT-PCR, respectively. Transfer of harbored or new infectious virions from TERT-2 cells was determined by co-culture with PBMCs. To bypass receptor-mediated entry and determine whether HIV-1 specific proteins could be translated, HIV-LTR regulated EGFP expression resulting from VSV-G pseudovirus infection was analyzed by flow cytometry. Results: Among putative HIV-1 receptors/co-receptors, TERT-2 cells expressed only heparin sulfate proteoglycans and CXCR4. APOBEC3G-, APOBEC3F-, Murr1-, Trim5á-, SLPI-, and hBD2-specific mRNAs were expressed, but appeared unaffected by HIV-1. When compared to permissive TZM-bl cells, TERT-2 cells expressed more APOBEC3G and 3F transcripts. TERT-2 cell-associated virions trans infect PBMCs for up to 48h post-incubation and TERT-2 nuclei contained integrated HIV-1 DNA, but HIV-1 specific mRNA transcripts were not detectable. After VSV-G pseudovirus infection, EGFP was expressed from the HIV-1 LTR promoter. In TERT-2/CD4 cells, HIV-IIIb internalization was increased, HIV-BaL was unaffected, and new transcripts and new virions were undetectable. Conclusion: HIV-1 infection in TERT-2 cells is restricted at viral entry by absence of CD4. Post-entry, APOBEC3G and 3F are candidate restriction factors, which persist when CD4 is expressed. Since TERT-2 cells trans infect PBMCs, the oral mucosa may serve as a non-permissive focus of primary HIV-1 infection.

Supported by NIH/NIDCR DE15056 and DE15503.