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), especially HPV types 16 and 18, are a major cause of cancer in women worldwide. HPV16, like most genital HPV types, relies on heparan sulfate proteoglycans (HSPGs) to attach to host cells and to the extracellular matrix. Attachment is mediated by surface-exposed basic residues of the major capsid protein, L1. This triggers conformational changes affecting L1 and the minor capsid protein, L2. However, it is not known what interaction triggers these structural changes and if any host cell protein is involved. Now we have identified a host cell chaperone, Cyclophilin B (CyPB), as essential for efficient HPV16 and HPV18 infection. CyPB, which is present on the cell surface in association with specific forms of O-sulfated HSPG as well as in the lumen of intracellular membrane structures, is an energy-independent enzyme, which catalyzes cis/trans isomerization of peptidyl-prolyl bonds. We demonstrate that CyPB facilitates conformational changes resulting in exposure of the L2 N-terminus, which is required for infectious entry. In addition, we present some evidence suggesting that members of the cyclophilin family are required for a second, probably intracellular, step of HPV16 infection. This is the first report implicating cell surface chaperones as essential host factors for viral infection.

Papillomaviruses are internalized via clathrin-dependent endocytosis. However, the mechanism by which viral genomes pass endosomal membranes has not been elucidated. In this report we show that the minor capsid protein L2 is required for egress of viral genomes from endosomes but not for initial uptake and uncoating and that a 23-amino-acid peptide at the C terminus of L2 is necessary for this function. Pseudogenomes encapsidated by L1 and L2 lacking this peptide accumulated in vesicular compartments similar to that observed with L1-only viral particles, and these mutant pseudoviruses were noninfectious. This L2 peptide displayed strong membrane-disrupting activity, induced cytolysis of bacteria and eukaryotic cells in a pH-dependent manner, and permeabilized cells after exogenous addition. Fusions between green fluorescent protein and the L2 peptide integrated into cellular membranes like the wild type but not like C-terminal mutants of L2. Our data indicate that the L2 C terminus facilitates escape of viral genomes from the endocytic compartment and that this feature is conserved among papillomaviruses. Furthermore, the characteristic of this peptide differs from the classical virus-encoded membrane-penetrating peptides.

Infection with various human papillomaviruses (HPVs) induces cervical cancers. Cell surface heparan sulfates (HS) have been shown to serve as primary attachment receptors, and molecules with structural similarity to cell surface HS, like heparin, function as competitive inhibitors of HPV infection. Here we demonstrate that the N , N ′-bisheteryl derivative of dispirotripiperazine, DSTP27, efficiently blocks papillomavirus infection by binding to HS moieties, with 50% inhibitory doses of up to 0.4 μg/ml. In contrast to short-term inhibitory effects of heparin, pretreatment of cells with DSTP27 significantly reduced HPV infection for more than 30 h. Using DSTP27 and heparinase, we furthermore demonstrate that HS moieties, rather than laminin 5, present in the extracellular matrix (ECM) secreted by keratinocytes are essential for infectious transfer of ECM-bound virions to cells. Prior binding to ECM components, especially HS, partially alleviated the requirement for cell surface HS. DSTP27 blocks infection by cell-bound virions by feeding into a noninfectious entry pathway. Under these conditions, virus colocalized with HS moieties in endocytic vesicles. Similarly, postattachment treatment of cells with heparinase, cytochalasin D, or neutralizing antibodies resulted in uptake of virions without infection, indicating that deviation into a noninfectious entry pathway is a major mode of postattachment neutralization. In untreated cells, initial colocalization of virions with HS on the cell surface and in endocytic vesicles was lost with time. Our data suggest that initial attachment of HPV to HS proteoglycans (HSPGs) must be followed by secondary interaction with additional HS side chains and transfer to a non-HSPG receptor for successful infection.

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.

Papillomaviruses are epitheliotropic non‐enveloped double‐stranded DNA viruses, whose replication is strictly dependent on the terminally differentiating tissue of the epidermis. They induce self‐limiting benign tumors of skin and mucosa, which may progress to malignancy (e.g. cervical carcinoma). Prior to entry into basal cells, virions attach to heparan sulfate moieties of the basement membrane. This triggers conformational changes, which affect both capsid proteins, L1 and L2, and such changes are a prerequisite for interaction with the elusive uptake receptor. These processes are very slow, resulting in an uptake half‐time of up to 14 h. This minireview summarizes recent advances in our understanding of cell surface events, internalization and the subsequent intracellular trafficking of papillomaviruses.

Human 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.