Bovine Papillomavirus Type 1: from Clathrin to Caveolin

Papillomaviruses (PVs) are nonenveloped double-stranded DNA viruses with an 8-kb circular genome. These viruses infect the basal, undifferentiated cells of the squamous epithelia and require host cell factors to replicate (41). PVs are responsible for causing disease in both animals and humans, infecting 6.2 million individuals in the United States each year (www.cdc.gov). Infection with one of the 13 high-risk oncogenic human PVs has been linked to cancers of the cervix, anus, mouth, skin, larynx, penis, and vulva (2, 3, 9, 13-15). Bovine PV (BPV) has served as an excellent model for the study of human PVs, and since BPV infection causes substantial morbidity in cattle, its study is merited (8). The current understanding of how these viruses enter and traffic through the cell has not been fully elucidated, and many discrepancies remain. The major capsid protein, L1, is responsible for the primary binding to the keratinocyte surface (24, 34). While a common cellular receptor has not been identified, two have been suggested: heparan sulfate (35) and the α6β4 integrin complex (16). The existing model of viral internalization supports the clathrin-mediated entry of BPV type 1 (BPV1), human PV type 16 (HPV16), and HPV58, despite a half-life of entry of 4 h (5, 12) and the observed neutralization of PV infection up to 24 h postattachment (10). The kinetics of particle entry by clathrin-mediated endocytosis are usually much faster, ranging from 5 to 15 min (17). Ligands taken up by clathrin-mediated endocytosis are internalized in clathrin-coated vesicles, which normally fuse to early endosomes and are sorted to either the late endosome/lysosome or the recycling endosome for transport back to the plasma membrane (37). HPV31 has been found by some investigators to utilize caveola-dependent endocytosis (5, 39), a pathway which involves the invagination of the plasma membrane at caveolin-stabilized lipid rafts and subsequent fusion with larger cholesterol-rich caveosomes (28). Another study recently demonstrated that HPV31 enters both human and monkey cells in a clathrin-mediated manner, with no reliance on the caveolar pathway (20). The ambiguity in the literature on PV entry suggests that viral entry is a complex process. A naked double-stranded DNA virus similar in structure to PV and previously grouped in the same family, JC virus (JCV), was shown to enter cells by clathrin-mediated endocytosis and then be escorted to a caveosome prior to delivery to the endoplasmic reticulum (ER) (32). It had been presumed that JCV trafficked solely via the clathrin-dependent pathway since viral entry was blocked by inhibitors of clathrin-dependent endocytosis and dominant negative Eps15 (30). Despite requiring caveolin-1 for infection, JCV infection was shown to be unaffected by chemical inhibitors of caveolar endocytosis, and the virus colocalized with transferrin (Tfn) at 20 min postinternalization (31). The transit of JCV from endosomes to caveosomes provides evidence that viruses can take advantage of the cross talk that occurs between cellular vesicles of different pathways. It appears that a lack of strict dependence on one pathway is an emerging theme in viral entry. Influenza virus, once thought to enter cells only by the clathrin-dependent pathway, was found to have an alternate means of entry (38). Blocking both the clathrin-dependent and caveolar pathways still resulted in infection, indicating that the virus could enter cells by an unknown route (38). The nonenveloped simian virus 40 (SV40) is noteworthy as one of the few viruses to utilize the caveola-dependent pathway of entry (29). Interestingly, SV40-activated endocytosis from the plasma membrane to the ER was demonstrated in a pathway not involving caveolin or clathrin (11). Another nonenveloped virus previously thought to enter by the clathrin-dependent pathway, poliovirus, has now been shown to enter by a mechanism that is tyrosine kinase and actin dependent. The pathway for poliovirus entry is clathrin, caveolin, flotillin, and microtubule independent (6). Our laboratory identified BPV1 pseudovirions (PsVs) in the ER using transmission electron microscopy, and immunofluorescence studies showed colocalization between BPV1 and the ER marker calnexin (4, 22). We characterized the interaction between the ER-resident protein syntaxin 18 and the BPV1 minor capsid protein L2, an interaction necessary for infection (22). Cells transfected with FLAG-tagged dominant negative syntaxin 18 prior to BPV1 infection were incapable of supporting a productive viral infection (4). The required interaction of BPV1 with the ER-resident protein syntaxin 18 for infection seemed to indicate that the observed viral particles in the ER were indeed part of the infectious pathway. The clathrin-dependent pathway, currently the accepted model of BPV1 entry, does not typically lead to the ER (27, 28, 37). A high particle-to-infectivity ratio has complicated studies of the infectious pathway of both PVs and polyomaviruses. Using polyomavirus-like particles encapsidating reporter DNA, investigators previously found two pathways for viral trafficking, one leading to the efficient delivery of the pseudogenome and the other along a noninfectious route (21). Our laboratory has described the internalization and trafficking of noninfectious mutant PsVs, which appear to traffic identically to wild-type (WT) PsVs until about 4 h postinternalization, when the WT virions arrive at the syntaxin 18-positive region of the cell and the mutant virions appear to be retained in the lysosomes (22). Indeed, it has been shown that for roughly every 2,000 wart-derived BPV1 virions, only 1 viral particle is infectious (33). It is difficult to differentiate between viruses that will lead to productive infection and those that will not, making dominant negative proteins and short hairpin RNAs (shRNAs) that block trafficking at specific points an especially useful tool for identifying the infectious pathway of a virus. In this paper, we used inhibitors of endocytosis, dominant negative caveolin-1, shRNA against caveolin-1, and a caveolin-1 scaffolding mutant (SM) to determine whether caveolin-1 was mediating the entry and trafficking of BPV1. We demonstrate that BPV1 is fully capable of infecting cells without cholesterol and caveolin-1 at the plasma membrane, but we show a nearly complete loss of infection when caveolin-1 expression is knocked down. This finding was of particular interest since BPV1 dependence on clathrin-mediated endocytosis has been well documented (12, 39). Our studies provide further evidence of the existence of cross talk between different types of endocytosis.