Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH.

Summary Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell-binding. Here, we analyze the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell-binding receptors for HPV-16, heparin-preincubated virus bound to the extracellular matrix (ECM) via laminin-332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S-domains of heparan sulfate (HS) chains of HSPGs, allowed HPV-16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope-specific antibody to the viral capsid after heparin-binding suggested that initial conformational changes in the HPV-16 virion occur during infection by interaction with ‘heparin-like’ domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV-16 infection.

Human papillomavirus type 18 (HPV18), one of the HPVs with malignant potential, enters cells by an unknown endocytic mechanism. The key cellular requirements for HPV18 endocytosis were tested in comparison to those for HPV16 and -31 endocytoses. HPV18 (like HPV16 and -31) entry was independent of clathrin, caveolin, dynamin, and lipid rafts but required actin polymerization and tetraspanin CD151, and the viruses were routed to the same LAMP-1-positive compartment. Hence, the viruses shared similar cellular requirements for endocytic entry. The human papillomaviruses (HPVs) are a family of nonenveloped DNA viruses with transforming potential. A particular subgroup of HPVs, the high-risk types, are additionally associated with cervical and anogenital cancers. Of those high-risk types, HPV type 16 (HPV16) is the most prevalent and the best studied. HPVs enter the basal cells of squamous epithelia by endocytosis during initial infection (1). For cell entry, most viruses employ one of several existing pathways for endocytosis (2, 3). These pathways are typically differentiated by the cellular proteins mediating endocytic vacuole formation, by the ultrastructural morphology of endocytic pits, and by the timing of vesicle formation (2-4). Although HPV16 cell entry was originally attributed to clathrinmediated endocytosis (CME) in various cell types (5-8), it has recently been shown that HPV16 enters cells by a novel ligandinduced endocytic pathway that is clathrin, caveolin, lipid raft, and dynamin independent but that depends on highly regulated actin dynamics and an association with CD151-containing tetraspanin-enriched microdomains (9-11) The high-risk-type HPV31 may differ in the mode of endocytosis from HPV16, as it has been described to enter into human keratinocytes by a caveolin/lipid raft-mediated mechanism (12). However, in COS-7 cells and 293TT cells, HPV16 and -31 endocytoses appear to occur by a similar mechanism that is independent of lipid rafts (5).One of the more-prevalent high-risk types, which has not been analyzed for its mode of endocytic entry, is HPV18. Here, we aimed to delineate the general endocytic requirements for infectious HPV18 endocytosis. In particular, we aimed to analyze whether HPV18 exhibits requirements similar to those of HPV16 or -31 for endocytic entry into host cells. Therefore, we compared the efficacy of cell entry after cell perturbation for the main determinants of several endocytic pathways in HeLa and HaCaT cells. The cell biological determinants that allow differentiation of the different endocytic pathways include clathrin for CME, caveolin for caveolin-mediated mechanisms, cholesterol depletion for lipid raft-dependent mechanisms (e.g., caveolar/lipid raft endocytosis or glycosphingolipid-enriched endocytic carriers), dynamin-2 for dynamin-mediated endocytic processes (e.g., CME, caveolar endocytosis, and the interleukin-2 pathway), and actin polymerization dynamics for actin-mediated endocytic processes (macropinocytosis, caveolar endocytosis, and HPV16 endocytosis) (...

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