Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV–induced diseases.

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex.In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.HPV entry | vesicular transport | mitosis | digitonin | nuclear vesicle A major hurdle of DNA viruses during a primary infection is successful navigation of the cytoplasm to deliver the viral genome to the nucleus. Foreign DNA that enters the cytoplasm is susceptible to being sensed by innate immune sensors (1). Not surprisingly, viruses have evolved mechanisms to evade detection by these sensors. For example, herpesviruses and adenoviruses both egress into the cytoplasm, yet protect their viral DNA by keeping it encased in viral capsids until directly transferring it into the nucleus through the nuclear pore complex. In contrast, the capsid is unlikely to protect papillomavirus (PV) genomes while traversing the cytoplasm, because the major capsid protein is lost in the endocytic compartment (2).The PV capsid is composed of two viral proteins, the major capsid protein L1 and the minor capsid protein L2, which enclose a chromatinized, circular, double-stranded DNA genome ∼8 kb in size (3-6). Following primary attachment and internalization, acidification of early endosomes triggers capsid disassembly (7-22). Host cell cyclophilins release the majority of L1 from the L2 protein, which remains in complex with the viral genome (2,23,24). A large portion of the L2 protein translocates across the endocytic membrane to engage factors, including the retromer complex, dynein, sorting nexins, and rab GTPases, that mediate transport to the trans-Golgi network (TGN) (25)(26)(27)(28)(29)(30)(31)(32)(33)). An siRNA screen has suggested that nuclear pore complexes are not required for nuclear entry, but that nuclear envelope breakdown during mitosis is necessary (34, 35).Currently, when and how the human PV (HPV) genome egresses from the membranous compartment is unclear. Here we present evidence indicating that after the onset of mitosis, the viral genome of HPV type 16 (HPV16), an HPV type...

dHuman papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.H uman papillomaviruses (HPVs) are small, nonenveloped epitheliotropic DNA viruses. HPV infection usually induces benign papillomas of the skin and mucosa. However, certain species, especially HPV16, are known as "high risk" due to their involvement in the progression to invasive carcinomas. Infection by HPV is considered necessary, though not sufficient, for the development of cervical cancer (1, 2). HPV infection is also associated with various anogenital and head and neck cancers (3). Despite the clear medical importance of preventing HPV-induced lesions, limited molecular detail regarding the attachment and entry of the virus is available. HPVs productively infect only epithelial cells in the skin and mucosa and depend on the differentiation of these cells for the completion of the viral life cycle (4). To bypass this obstacle, an in vitro surrogate system for viral propagation using a marker gene encapsidated into the viral capsid proteins was developed (5-7). This pseudovirus system overcomes the tropism and species specificity for viral propagation displayed by HPVs, allowing for study of the early events in the infection process.The papillomavirus virion is composed of the major and minor capsid proteins, L1 and L2, respectively. L1 is present in 360 copies organized into 72 pentamers, referred to as capsomeres (8-10). The L2 protein is present in an undetermined number of copies and is initially hidden inside the L1 structure prior to attachment to the cell surface (11). The outer virion shell, formed via pentavalent and hexavalent capsomere interactions between L1 molecules, medi...

High risk human papillomavirus types 16 (HPV16) and 18 (HPV18) can cause cervical cancer. Efficient infection by HPV16 and HPV18 pseudovirions requires interactions of particles with cell-surface receptor heparan sulfate oligosaccharide. To understand the virus-receptor interactions for HPV infection, we determined the crystal structures of HPV16 and HPV18 capsids bound to the oligosaccharide receptor fragment using oligomeric heparin. The HPV-heparin structures revealed multiple binding sites for the highly negatively charged oligosaccharide fragment on the capsid surface, which is different from previously reported virus-receptor interactions in which a single type of binding pocket is present for a particular receptor. We performed structure-guided mutagenesis to generate mutant viruses, and cell binding and infectivity assays demonstrated the functional role of viral residues involved in heparin binding. These results provide a basis for understanding virus-heparan sulfate receptor interactions critical for HPV infection and for the potential development of inhibitors against HPV infection. Human papillomaviruses (HPVs)4 are non-enveloped small DNA viruses of great medical importance. Among the large group of HPVs known by now, sexually transmitted genital high risk HPV types are the cause for the development of a variety of epithelial tumors, especially cervical carcinoma (1). Cervical cancer is the second leading cause of death among female cancer patients worldwide. HPV16 and HPV18 stand out, as they are causally linked to Ͼ70% of cervical cancer cases (2).HPV particles consist of 72 pentamers of the major capsid protein L1, which forms the virus outer shell and encapsidates the viral DNA (3, 4). The minor capsid protein L2 is present at up to 72 copies and is hidden inside the capsid with exception of a small N-terminal section (5, 6). Efficient infection by HPV16 and HPV18 pseudoviruses requires the interactions of the L1 protein with extracellular matrix (ECM)-and cell surface-resident heparan sulfate receptor in vitro (7-9) as well as in vivo models (10). Homologs of heparan sulfate polysaccharide or heparin, secreted by mast cells, can inhibit HPV infection (7-9).Cell-surface heparan sulfates are linear and highly negatively charged oligosaccharides that are covalently linked to proteins. They can serve as the attachment receptors for several important human virus pathogens (7,11,12). Despite considerable efforts, the interactions between HPV and the heparan sulfate oligosaccharides that initiate infection are poorly understood. Here, we determined the co-crystal structure of HPV16 and HPV18 capsids bound to oligomeric heparin. We found that the highly negatively charged heparin fragment binds to multiple locations on the capsid surface mainly through charge-charge interactions. On the basis of the structure, we generated mutant virus to disrupt the interactions with heparin. ECM and cell binding assays combined with infectivity measurements showed that substitution of key HPV residues involved in bi...