Human Papillomavirus Type 16 Infection of Human Keratinocytes Requires Clathrin and Caveolin-1 and Is Brefeldin A Sensitive

Human papillomavirus type 16 (HPV16) has been identified as being the most common etiological agent leading to cervical cancer. Despite having a clear understanding of the role of HPV16 in oncogenesis, details of how HPV16 traffics during infection are poorly understood. HPV16 has been determined to enter via clathrin-mediated endocytosis, but the subsequent steps of HPV16 infection remain unclear. There is emerging evidence that several viruses take advantage of cross talk between routes of endocytosis. Specifically, JCV and bovine papillomavirus type 1 have been shown to enter cells by clathrin-dependent endocytosis and then require caveolin-1-mediated trafficking for infection. In this paper, we show that HPV16 is dependent on caveolin-1 after clathrin-mediated endocytosis. We provide evidence for the first time that HPV16 infection is dependent on trafficking to the endoplasmic reticulum (ER). This novel trafficking may explain the requirement for the caveolar pathway in HPV16 infection because clathrin-mediated endocytosis typically does not lead to the ER. Our data indicate that the infectious route for HPV16 following clathrin-mediated entry is caveolin-1 and COPI dependent. An understanding of the steps involved in HPV16 sorting and trafficking opens up the possibility of developing novel approaches to interfere with HPV16 infection and reduce the burden of papillomavirus diseases including cervical cancer.Human papillomavirus (PV) type 16 (HPV16) is a member of the family Papillomaviridae, a group of double-stranded DNA (dsDNA) viruses with a tropism for squamous epithelia (70). Most PV infections result in benign lesions, although a subset of high-risk HPVs are capable of malignant transformation, resulting in various cancers including cervical carcinoma (21, 38). Infection with HPV16 is responsible for causing approximately half of the cases of invasive cervical cancer (7). In spite of the link between HPV16 and cervical cancer, the intracellular movement of HPV16 through target keratinocyte cells during infection has not been defined in detail.Viruses can enter into target cells by taking advantage of the cell''s natural endocytosis machinery (60). One of the best-characterized modes of internalization is by receptor-mediated, clathrin-dependent endocytosis. In this mode of entry, clathrin-coated pits internalize cargo into clathrin-coated vesicles, which are pinched from the plasma membrane by dynamin-2 in order to internalize (68). The process of clathrin-mediated endocytosis occurs rapidly, resulting in the delivery of cargo to early/sorting endosomes within seconds to minutes (23, 31). From the sorting endosome, most clathrin-dependent ligands are trafficked back to the plasma membrane in recycling endosomes or to lysosomes for degradation (35, 56). Another well-studied model of ligand entry is caveolin-1-mediated endocytosis. The caveolar pathway typically involves entry via cholesterol-rich caveolae at the plasma membrane, which deliver their contents to pH-neutral organelles known as caveosomes (44, 65). The delivery of cargo from caveosomes to the Golgi apparatus and the endoplasmic reticulum (ER) was demonstrated previously (44, 46, 50). The traffickings of cargo internalized via clathrin- and caveolin-1-mediated endocytosis were once thought to be separate; however, it is becoming evident that viruses including bovine PV type 1 (BPV1), JCV, HPV31, and BKV rely on both pathways depending on the stage of infection (29, 32, 50, 63).PV internalization is preceded by virion attachment to the extracellular matrix, followed by binding to heparan sulfate (14, 15, 25). The involvement of a secondary receptor has been suggested, putatively an alpha-6 integrin (24, 37). Postbinding, a conformational change in the PV capsid results in a furin cleavage event at the N terminus of the minor capsid protein L2, which has been suggested to play a role in the endosomal escape of the viral genome (19, 30, 52). An increasing body of evidence supports the entry of HPV16 by clathrin-mediated endocytosis (9, 27, 62). Electron microscopy of HPV16 infection in COS-7 cells demonstrated HPV16 pseudovirions in clathrin-coated vesicles 20 min after entry and within structures resembling endosomes by 1 h postentry (9). HPV16 infection of HaCaT keratinocyte, COS-7, and 293TT cells has been blocked by chlorpromazine, an inhibitor of the formation of clathrin-coated pits (9, 27, 62, 67). Importantly, those studies showed that two inhibitors of caveolin-1-mediated internalization, filipin and nystatin, did not interfere with HPV16 infection (9, 27, 62). Our laboratory demonstrated the importance of dynamin in HPV16 infection, presumably in the scission of clathrin-coated vesicles from the plasma membrane (1). Recently, a clathrin-, caveolin-, and dynamin-independent endocytosis of HPV16 was suggested, although the use of the HPV18-positive, heteroploid HeLa cell line calls into question the relevance of this finding to natural infection (64).In a previous study, we described the postentry trafficking of BPV1 from endosomes to caveolin-1-positive vesicles, similarly to a related nonenveloped dsDNA virus, JCV (32, 50). Our data demonstrated that the infectious route of BPV1 involved entry by clathrin-mediated endocytosis followed by transport to the caveolar pathway in order to traffic to the ER (32). We found that BPV1 infection was neutralized by an antibody that prevented viral particle transport to the ER (33). The movement of BPV1 from the endosome to the caveosome provides a possible explanation for why BPV1 trafficking is so slow compared to those of other ligands of clathrin-mediated endocytosis (20, 26). The kinetics of BPV1 and HPV16 entry were previously reported to be identical, and the coincident internalization of HPV16 and BPV1 virus-like particles (VLPs) showed colocalization between the VLPs during infection (20, 62). These data suggest that HPV16 and BPV1 infection may be occurring by a similar mechanism.Our goal in the present study was to determine the intracellular trafficking events leading to HPV16 infection. The use of reporter virion technology has allowed the production of high-titer HPV16 virions by a method previously shown to yield virions that are infectious in vivo (16). In this study, we used HPV16 reporter virions to study HPV16 infection in the spontaneously immortalized human HaCaT keratinocyte cell line. Our data show that the infectious route of HPV16 is from early endosomes to caveolin-1-positive vesicles and then to the ER. Using immunofluorescence and short hairpin RNA (shRNA) against caveolin-1, we demonstrate the importance of the caveolar pathway after HPV16 has been internalized. We show that HPV16 infection was blocked by inhibiting the formation of COPI transport vesicles, which function in trafficking between the ER and the Golgi apparatus and from caveosomes to the ER (5, 39). We provide evidence that after reaching the caveosome, HPV16 requires passage to the ER for successful infection, a trafficking event made possible by COPI vesicle-mediated movement from the caveosome to the ER.