BKV nephritis has become a severe problem for renal transplant patients, with about half of the cases of BKV nephritis becoming graft losses. However, there are few in vitro studies of BKV infection and only three reports of in vitro BKV infection of HRPTEC (1
). It seems that the nature of the host cells has a profound effect upon the efficiency and consequences of viral infection. Therefore, it is imperative to use HRPTEC, one of the main natural targets of BKV infection, in order to elucidate the exact mechanism of BKV nephritis in humans. Low et al. reported that BKV actually infected HRPTEC in vitro, as detected by the expression of high levels of T-Ag, VP1, and viral DNA 36 h after infection (24
). The role of caveolae in BKV internalization in human cells was not addressed in detail.
Though it is still controversial whether caveolae play a role in endocytosis, SV40 has been reported to internalize through caveolae (4
) and BKV has also been reported to internalize into HRPTEC through flask-like invaginations similar to caveolae, as shown by morphological analysis (8
). Moreover, previous reports indicated that N-linked glycoprotein with α(2,3)-linked sialic acid served as a BKV receptor in monkey-derived Vero cells (9
), and gangliosides GD1b and GT1b were also reported to work as receptors for BKV in human prostate carcinoma cells (25
). Since these gangliosides seem to be strongly associated with caveolae, it is possible that caveolae also play a role in endocytosis for BKV.
At first, we focused on establishing an experimental protocol for incubating HRPTEC with BKV. Low et al. reported that high levels of T-Ag were expressed at 36 h when HRPTEC were infected with BKV at an MOI of 5 FFU/cell (24
). Our results showed the highest percentage of BKV infection at 72 h when HRPTEC were infected with BKV at an MOI of 0.5 FFU/cell. As this MOI was lower than that utilized by Low et al., the apparent infection timing may have been longer than what was reported previously (24
We then examined the role of cholesterol in BKV internalization by using the cholesterol-depleting agents MBCD and Nys. Our results suggested that cholesterol was required for BKV infection of HRPTEC, because MBCD or Nys caused inhibition of BKV infection in HRPTEC and they did not interfere with the internalization of transferrin, which is a marker of clathrin endocytosis. These results indicated that BKV entered HRPTEC through structures in the cell membrane that are enriched in cholesterol, such as caveolae, and these agents had no effect upon the clathrin-mediated pathway. Damm et al. (5
) and Pelkmans et al. (31
) reported that a combination of Nys and progesterone blocked SV40 infection of a human hepatoma cell line (HuH7 cells) and CV-1 cells. Gilbert et al. reported that MBCD or Nys inhibited SV40 infection of murine fibroblasts (NIH 3T3 cells) and primary baby mouse kidney epithelial cells, and also reported that MBCD or Nys inhibited mPy infection against GD1a-supplemented rat glioma cells (C6 cells) (16
). These results implied that SV40 and mPy entered cells through the caveolae. Moreover, BKV infection of monkey-derived Vero cells was reported to be inhibited by MBCD (12
), providing additional support for our conclusion. Thus, our experiments strongly support a role for cholesterol in BKV internalization into cells.
Caveolins are scaffolding domains of caveolae. Three caveolins exist: caveolin-1, caveolin-2, and caveolin-3. Caveolin-1 and -2 are coexpressed in most cells; caveolin-3 is expressed in muscle-specific cells, such as skeletal, cardiac, and smooth muscle cells. Caveolin-1 expression can induce the formation of caveolae, but caveolin-2 cannot. Therefore, disruption of the caveolin-1 gene results in the abolishment of caveolae (22
). Our results showed that siRNA-mediated Cav-1 knockout caused inhibition of BKV infection in HRPTEC, but clathrin knockout did not. It seems likely that BKV enters HRPTEC through caveolae and not through the clathrin-mediated pathway. Eash et al. reported that BKV infection of Vero cells was inhibited by the expression of a dominant-negative Cav-1 mutant compared to the infection of Vero cells expressing wild-type Cav-1. Despite the different host cells, our data are in good concordance with their results (12
). We also investigated the dynamics of the colocalization of labeled BKV with Cav-1. Labeled BKV particles were colocalized with Cav-1, but not with clathrin, and the colocalization rate of labeled BKV with Cav-1 peaked at 4 h after incubation. Our data suggest that BKV entered HRPTEC through caveolae and that, at about 4 h after infection, BKV particles were mostly trapped in the caveolae. Pelkmans et al. (30
) and Gilbert and Benjamin (15
) reported colocalization of polyomaviruses SV40 and mPy with Cav-1. These viruses were frequently trapped in caveolae until 1 h after incubation. Our data suggest that the internalization of BKV proceeds relatively slowly.
To understand more precisely the mechanisms and kinetics of BKV infection, it is important to employ HRPTEC, which are the main target of BKV infection in the actual clinical situation. It must be taken into consideration that the viral life cycles are quite different in each cell type. For example, SV40 was reported to be unable to infect HRPTEC (24
), but was efficient in the infection of monkey tubular epithelial cells (16
), HuH7 cells (5
), NIH 3T3 cells (16
), and CV-1 cells (30
). mPy was reported not to enter NIH 3T3 cells and baby mouse kidney epithelial cells via caveolae (16
), in contrast to normal murine mammary gland epithelial cells (35
). These differences seem to come from the inherent specificities of the polyomaviruses for each of the different cell lines.
In summary, our results show that depletion of cholesterol and disruption of Cav-1 mRNA inhibit BKV infection of HRPTEC, and labeled BKV particles were colocalized with Cav-1. Considering that Cav-1 is a scaffolding protein of caveolae and cholesterol is abundant in caveolae, our results suggest that BKV enters HRPTEC through caveolae in vitro. Moreover, our results indicate that BKV are trapped in caveolae most frequently at 4 h after infection. BKV infection is relatively slow in comparison to mPy and SV40 infection.