HIV infection of LNCaP cells. LNCaP or HeLa-CD4 cells in 24-well culture plates (10⁴ cells/well) were infected with HIV either with or without gp120 protein on its envelope [Env(-) and Env(+) HIV]. A) A significantly high percentage of EGFP-positive cells was demonstrated in the LNCaP cell cultures infected by HIV Env(-) virus (9–14%). Infection of HeLa-CD4 cells by HIV Env(+) was used as a positive control to assess anti-gp120 or -gp160 function of the antibodies. Infection of HeLa cells was performed as a negative control. The infections of HIV either with or without Env showed very low infectivity for HeLa cells, as demonstrated with no EGFP-positive cells in the infected culture, or occasionally there were one or two EGFP-positive colonies. In other experiments, we also infected HeLa-CD4 cells with Env(-) HIV_NL4-3, and found that the Env(-) HIV strain did not infect HeLa-CD4. These results have been previously reported (22). B) Infection of CD4(-) cell lines by HIV_NL4-3-Env(-)-EGFP virus prepared from an oral epithelial cell line derived from a patient. The cell line was established and maintained in our laboratory. C) Infection of the LNCaP and HeLa-CD4 cell lines by HIV at various concentrations. Because the figure is in log scale, the standard deviations do not appear clearly. These are: 1) LNCaP: 900 ± 38 (5 ng), 205 ± 11(1.5 ng), 24 ± 5.6 (0.5 ng), 8.5 ± 0.7 (0.15 ng), 2.5 ± 0.7 (50 pg); and 2) HeLa-CD4: 1114 ± 115 (5 ng), 638 ± 47 (1.5 ng), 80 ± 10 (0.5 ng), 14.5 ± 2.1 (0.15 ng), 3.5 ± 0.71 (50 pg), 1 ± 0 (15 pg).

Inhibition of HIV infection by antibodies specific to CLDN-7. A) Immunostaining of CLDN-7²¹¹ (upper panel) and CLDN-7¹⁵⁸ (lower panel) by CLDN-7-specific polyclonal antibodies. 293T cells (5 × 10⁴) were plated into 35-mm plates 24 hours prior to transfection. CLDN-7 plasmids (3 μg) were used for transfection of each 35-mm plate. At two days post-transfection, the cells were immunostained. CLDN-7 antibodies were added into the plates overnight at 4°C. B) Antisera from NIH (anti-gp160, cat. 191), Zymed (anti-CLDN-7, 0.25 mg/ml), or made by us were added to LNCaP cell cultures at 1:100 v/v 10 minutes prior to adding HIV. Six days post-infection, EGFP-positive cells were counted. Infection of LNCaP cultures with no antibodies was set as the control. EGFP-positive cells in the culture treated with antibodies against CLDN-7 demonstrated 27% ± 0.6% viral infection compared to the control with no antibodies in the cell culture.

Expression of CLND-7²¹¹ in PBL and macrophages. RNA isolated from unstimulated PBL, interleukin 2-stimulated PBL, macrophages, and control cell lines was analyzed using RT-PCR. RNA samples were reverse transcribed using the oligo-dT primer, followed by PCR using the primers 5'-CTCCTCTGACTTCAACAGCG-3' and 5'-TGTTGCTGTAGCCAAATTCG-3' to detect the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene as RNA standard, and the primers described previously [26] for detecting CLDN-7 RNA. Panels A and B are RT-PCR from different samples.

Two models for gp120-independent HIV infection. Model 1: HIV may use cellular proteins that are anchored to its envelope to bind to either CLDN-7 or other proteins. In LNCaP, both CLDN-7 and associated proteins involved in gp120-independent infection are present. In 293T cells, neither CLDN-7 nor the other infection-related membrane protein(s) is present. Expression of CLDN-7 in 293T may increase infectivity, but the levels of infection of the CLDN-7-modified 293T may still be significantly lower than those for LNCaP. Model 2: HIV may use cellular proteins that are anchored to its envelope to bind to a CLDN-7-associated complex. Although transfection of CLDN-7 can express this protein on the cell surface, the lack of the CLDN-7-associated protein decreases the binding of virus to the target cells.

Effect of CLDN-7 molecules upon infection of 293T cells by HIV_NL4-3. A) HIV_NL4-3 Env(-) virus infection of either CLDN-7²¹¹- or CLDN-7¹⁵⁸-modified 293T cells. In experiment 1, HIV Env(-) at 50 ng of p24 was used to infect 10⁴ cells in each well; in experiment 2, 10⁴ cells were infected by virus at 100 ng of p24. B) HIV_NL4-3 Env(+) virus infection of either CLDN-7²¹¹- or CLDN-7¹⁵⁸-modified 293T cells. Plasmid pCDNA3-transfected 293T cells were also used as a control.

Infectivity of CLDN-7²¹¹- or CLDN-7¹⁵⁸-modified Env(-) virus. A) Western blot of proteins isolated from the virus generated from the transfected 293T cells by pNL4-3-EGFP-Env(-) and CLDN-7. Cell culture medium collected from cells transfected with CLDN-7 was used as a control to assess the background of CLDN-7 in microvesicles. Cellular proteins isolated from CLDN-7 plasmid-transfected cells were used as a positive control. The monomers of CLDN-7 are approximately 22 kd. Lane M is a molecular marker lane. The monomers of CLDN-7 are approximately 22 kd. B) CLDN-7-modified HIV_NL4-3 Env(-) virus showed significantly higher infectivity for 293T cells. C) Infection of either LNCaP or CEM CD4(+) T-lymphocyte cell lines by CLDN-7-modified HIV, with approximately a 1.5- to 2-fold increase of viral infection. D) CLDN-7-modified HIV_JRCSF virus with intact gp120 showed significantly higher infectivity for a CD4(-) cell line, PC-3, than the virus with no CLDN-7 on its surface.

Expression of CLDN-7 molecules in human tissues. A nylon filter preloaded with RNA from various tissues from BD Clontech (Palo Alto, CA) was used to assess the expression levels of CLDN-7 in various tissues. The left panel shows the hybridization of the tissue samples in the filter by a CLDN-7 probe containing the coding region, and the right panel shows the corresponding tissues.