Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.Papillomaviruses are highly species-and tissue-specific viruses primarily found in higher vertebrates. They infect exclusively basal cells of epithelia and induce squamous epithelial and fibroepithelial tumors, e.g., warts (papillomas) and condylomata. To date, more than a hundred human papillomaviruses (HPVs) have been identified, and some of them are strongly associated with malignant epithelial lesions, particularly genital carcinoma (for a review, see reference 13). Infectious virions recovered from naturally occurring warts of rab-

BackgroundInfectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway.Methodology/Principal FindingsUsing immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16.Conclusions/SignificanceTetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

Due to the association of human papillomaviruses (HPV) with development of multiple carcinomas, especially cervical carcinomas, early diagnosis and prevention of infection with HPV are of great medical and economic importance. Knowledge of the early steps of papillomavirus infection, which results in infectious entry, will help develop means to prevent HPV-induced lesions. Since HPV are difficult to propagate in cell culture, surrogate infection systems with marker-encoding viral capsids, called HPV pseudovirions, have been developed and successfully used in investigating the HPV entry pathway as well as in testing of substances interfering with HPV infection (2, 33). These studies have led to the identification of specifically modified heparan sulfate proteoglycans (HSPGs) as primary attachment receptors for papillomaviruses (13,15) and to heparin and other sulfated polysaccharides as inhibitors of HPV infection (1, 7, 13). Recently, carrageenan, an unbranched sulfated polysaccharide from algae with saccharide linkages reminiscent of galactosaminoglycans, has been reported to inhibit HPV infection primarily by preventing the binding of virions to the cell (4). Dispirotripiperazine (DSTP) derivatives represent another substance class with proven antiviral potential. DSTP27 (an N,NЈ-bisheteryl derivative of DSTP), one of the most active derivatives of this new class of low-molecular-weight antiherpetic compounds, interacts with specific forms of cell surface HSPGs (26). In addition to the inhibition of herpes virus attachment and infection, DSTP27 efficiently blocks the attachment and uptake of members from other virus families that depend on HSPGs as primary attachment molecules (25). In contrast to the HS analogs such as heparin and pentosan polysulfate that have short-lived effects, pretreatment of cells with DSTP27 induces a longlasting antiviral effect. Based on computer modeling, DSTP27 possibly interacts with two O-sulfate groups located on neighboring saccharides of the HS chain (27). Using the octosaccharide essential for HS-mediated entry of herpes simplex virus type 1 (HSV-1) into host cells (20), these computational studies further show that DSTP27 may additionally interact with a carbonyl group, thus increasing the strength of compound binding.Since HPV bind specifically to sulfated polysaccharide residues of cell surface HSPGs, particularly 2-O-and 6-O-sulfated HS chains in addition to N-sulfated residues (27), DSTP27 was predicted to work as a potent inhibitor of HPV infection. In this report we demonstrate that DSTP27 efficiently prevents HPV infection when applied several hours pre-or postinfection of cells. This is achieved by two putatively different mech-* Corresponding author. Mailing address:

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 pHdependent 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.