To determine which key entry receptors are expressed on HCjE cells, RT-PCR, immunofluorescence, and flow cytometry were performed. RT-PCR results demonstrated that mRNA specific for nectin-1, nectin-2, HVEM, and 3-
OST-3 receptor genes exist in HCjE cells
(Fig. 5) . HeLa cells also expressed nectin-1, nectin-2, HVEM, and 3-
OST-3 mRNA, whereas CHO-K1 cells failed to show any HSV-1 entry receptor mRNA, as expected (data not shown). Next, to examine protein expression, laser-scanning spectrum confocal microscopy was used for immunofluorescence assay. We excluded nectin-2 from subsequent studies because it mediates the entry of some laboratory-generated mutants but not wild-type strains of HSV-1.
7 Receptor-specific primary antibodies combined with FITC-conjugated secondary antibodies were used. Immunofluorescence data shown in
Figure 6demonstrate that nectin-1, HVEM, and 3-
OS HS are expressed along the cell membrane of HCjE cells
(Fig. 6) . The control cells, with only incubation of secondary FITC-conjugated antibody, did not show any fluorescence on the cell membrane. To verify and estimate the relative levels of receptor expression, flow cytometry was used. Although the expression of all the three receptors was evident again, it was also apparent that nectin-1 was likely the most abundant
(Fig. 7a) , followed by HVEM and 3-
OS HS
(Figs. 7b 7c) . 3-
OS HS is a rare modification of HS, which may be responsible for the low expression of 3-
OS HS detected by flow cytometry.
12 To verify our receptor expression findings in vivo, immunohistochemistry was performed using sections of palpebral conjunctiva obtained from adult (8-month-old) female BALB/c mice. As shown
(Fig. 8) , strong nectin-1 expression was detected in the conjunctival epithelium and sebaceous glands with weak to modest staining in the lamina propria. Weak HVEM staining was detected in the conjunctival epithelium and sebaceous glands, and local HVEM staining was detected in the lamina propria. However, the weak staining may be attributable to poor reactivity of the rabbit HVEM antibody raised against human HVEM to the murine tissue. No clear signals were reported for 3-
OS HS (data not shown).