The recognition of the host cell by the virus is a key determinant of the efficiency of a viral infection. This initial interaction at the plasma membrane also can determine which route the virus will take to the nucleus. For polyomaviruses, this interaction leads to caveola-mediated entry in the cases of SV40, BKV, and Py or clathrin-mediated entry in the case of JCV (19
). The integrity of this initial stage of infection may be critical for correct conformational changes, unfolding, and eventual disassembly of the capsid. In this study, we report that BKV exhibits interactions at the plasma membrane similar to those reported for SV40 and Py, which use gangliosides as receptors, but different from that of JCV, which has been reported to use protein molecules as receptors.
Our experiments define GD1b and GT1b as molecules that bind BKV in vitro and as receptors for the virus on cells. The lack of binding of BKV to other gangliosides in our studies suggests that it is the carbohydrate moiety to which the virus binds rather than the identical ceramide lipid moiety present in all the gangliosides. While others have described sialic acid or gangliosides in general as potential receptors for BKV (44
), this is this first study in which specific molecules and moieties have been identified as receptors. We demonstrated, using a sucrose flotation assay, that BKV interacts with erythrocyte membranes. Through treatment with proteinase K and neuraminidase we determined that the interaction between BKV and the membranes was not dependent on proteins but instead was dependent on sialic acid residues, which are commonly found in gangliosides. We then demonstrated that BKV interacted specifically with the gangliosides GD1b and GT1b, but not GM1, when these gangliosides were incorporated into liposomes. Additionally, BKV was unable to infect LNCaP cells without the addition of GD1b or GT1b to the cells. After we obtained these results, it was also reported that while normal prostate epithelial cells express the gangliosides GD1b and GT1b, these gangliosides are absent on LNCaP cells (39
The interaction between BKV and the gangliosides GD1b and GT1b, but not GM1, leads us to conclude that a strong interaction between the virus and the ganglioside is dependent upon a disialic acid motif. This result suggests that the binding pocket on the BKV capsid is deeper than that of SV40, but possibly similar to that of Py, which also uses GT1b as a plasma membrane receptor (47
). Also interesting is that the two gangliosides which confer susceptibility to infection both contain a terminal α2-8-linked sialic acid residue. It is possible that this specific linkage is required for a productive interaction between the virus and the receptor.
We demonstrated that BKV interacts with ER membranes and that trafficking of BKV through the ER is critical for delivery of the viral genome to the nucleus, as evidenced by TAg expression. Intracellular pH changes are also necessary for efficient BKV infection (15
), but exactly where these changes take place remains to be determined. The standard route of transport during ganglioside synthesis is production of the ceramide lipid in the ER, addition of the specific sugar moieties in the Golgi apparatus, and transport of the completed ganglioside to the plasma membrane. These findings, when taken together with our results, suggest that a fraction of the gangliosides at the plasma membrane traffic back to the ER, and the BKV bound to these gangliosides follows a similar pathway. Previous studies demonstrating interactions of SV40 and Py with gangliosides support this model (18
), but it is also possible that any or all of these viruses interact with additional molecules present in the ER. While all three viruses enter the cell by a caveola-dependent mechanism, and exiting from the ER is a critical step, neither SV40 nor Py has been shown to colocalize with the Golgi apparatus (15
). It is possible that these viruses, as well as BKV, use a BFA-sensitive pathway that does not involve the Golgi apparatus, or that they pass too rapidly through the Golgi apparatus to be detected.
While BKV can infect a wide variety of cells in culture, in the human host there are three specific cell types in which the virus is commonly found: tonsillar lymphocytes, peripheral blood mononuclear cells, and kidney epithelial cells (22
). Gangliosides likely play a role in the infection of each of these cell types. The initial BKV infection of a child likely occurs in the tonsils, where GD1b and GT1b have both been observed (48
). BKV infection then progresses to peripheral blood mononuclear cells, from which these two gangliosides have also been extracted (50
). Finally, BKV remains subclinical for the lifetime of the host in the kidney, where it has been shown that GD1b and GT1b are present in both cortical tubular and medullary tissue (28
). The mechanism of transmission of BKV remains unknown, but it is not thought to be transmitted from mother to fetus. While pregnant women commonly secrete BKV in their urine, the activity of the enzyme responsible for the creation of the α2-8-linked sialic acid residue in both GD1b and GT1b is reduced nearly 80% in the tissue surrounding the fetus (52
). It is possible that the downregulation of GD1b and GT1b in these tissues provides a level of protection to the fetus from the virus reactivation that occurs during pregnancy.
Given the importance of BKV infection and reactivation in transplant patients, it is critical to understand the life cycle of the virus. The identification and characterization of BKV receptors and trafficking offer potential targets for pharmaceutical agents. Thus, further studies to elucidate interactions between BKV and its target cell types are required for a complete understanding of the mechanisms used by the virus to infect the host and cause disease.