Human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes

Abstract

Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.

FIG. 1.

(A) Quantitative PCR analysis of HIV-infected NHOKs and PBLs. NHOKs were exposed to 400 ng of live HIV-1_NL4-3 or heat-inactivated (HI) virus for 2 h at 37°C in the presence of 10 μg of Polybrene per ml. Cells were rinsed and cultured for 16 h prior to DNA extraction. Viral DNA was PCR amplified with the R/U5 primer pair (M667 and AA55, 140 bp) to detect early viral LTR reverse transcripts and LTR/gag (M667 and M661, 200 bp) for full-length reverse transcripts. The amplified products were resolved on a 6% nondenaturing polyacrylamide gel and visualized by autoradiography. Each lane represents DNA from approximately 5 × 10³ cells. β-Globin primers were used in parallel for a loading control. Heat inactivation of the virus was done for 1 h at 65°C, and activated PBLs were used as a positive control for virus infection. This is a representative result of more than five independent experiments. (B) Quantitative PCR analysis of NHOKs infected with primary HIV-1 strains.

FIG. 2.

(A) p24^gag antigen accumulation in the culture medium of HIV-1_NL4-3-infected NHOKs. The cell-free culture medium of HIV-infected NHOKs was removed at the times indicated for measurement of p24^gag levels by ELISA. The graph shows p24 levels versus the number of days postinfection, and the results are averages of three independent experiments. HI, heat-inactivated virus. (B) Experiment similar to that in panel A, except that the replication of HIV-1_NL4-3 was compared between NHOKs and PBLs by assaying p24 levels by the ELISA method. (C) Summary of p24 levels in T-lymphotropic NL4-3-, ELI-, dualtropic 89.6-, and CCR5-tropic Ba-L-infected NHOKs.

FIG. 3.

Expression of HIV receptors on NHOKs. After culture for 2 weeks, 10⁶ NHOKs were incubated with PE-conjugated MAbs (CD4, GalCer, CXCR4, and CCR5) and assayed by flow cytometry. Panels: A, Epithelium Pan control; B, CD4; C, CCR5; D, CXCR4; E, Galactocerebroside.

FIG. 4.

HIV-1_NL4-3 requires GalCer and/or CXCR4 for NHOK infection. NHOKs were pretreated with various HIV-1 entry inhibitors for 1 h at 37°C and then exposed to HIV-1_NL4-3 for 1 h at 37°C. The final concentrations of the inhibitors were 10 μg of recombinant soluble CD4 per ml, 100 ng of SDF-1α per ml, 50 μg of nonspecific MAb per ml, and 2.5 μg of anti-GalCer MAb per ml. Viral replication was analyzed by determining the p24 antigen concentration in the postinfection culture supernatant.

FIG. 5.

(A) Viral transmission from NHOKs to PBLs during coculture. NHOKs (5 × 10³) were incubated for 2 h in culture supernatants containing approximately 300 ng of HIV-1_NL4-3 per ml. Following incubation, NHOKs were washed extensively and cultured overnight with an equal number of uninfected activated PBLs. Nonadherent PBLs were then harvested, washed extensively, and incubated. After 48 h of culture, HIV-1 infection of cocultured PBLs was assessed by PCR amplification of viral early reverse transcripts. HI, heat inactivated. (B) HIV-1_NL4-3 replication in coculture assays. p24 levels were assayed by ELISA in supernatants from the final 48 h of PBL culture in the experiment shown in panel A. Triangles, p24 levels of originally infected NHOKs before coculture; squares, p24 levels of rescuing PBLs after overnight incubation with infected NHOKs. (C) Experiment parallel to those in panels A and B following exposure of NHOKs to 300 ng of HIV-1_Ba-L per ml for 2 h.