Luise Florin

Human Papillomavirus Infection Requires Cell Surface Heparan Sulfate

ABSTRACT 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.

Clathrin- and Caveolin-Independent Entry of Human Papillomavirus Type 16—Involvement of Tetraspanin-Enriched Microdomains (TEMs)

Background Infectious 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 Findings Using 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/Significance Tetraspanins 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.

A Membrane-Destabilizing Peptide in Capsid Protein L2 Is Required for Egress of Papillomavirus Genomes from Endosomes

ABSTRACT 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.

Inhibition of Transfer to Secondary Receptors by Heparan Sulfate-Binding Drug or Antibody Induces Noninfectious Uptake of Human Papillomavirus

ABSTRACT 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.

Identification of a Dynein Interacting Domain in the Papillomavirus Minor Capsid Protein L2

ABSTRACT Papillomaviruses enter cells via endocytosis (H. C. Selinka et al., Virology 299:279-287, 2002). After egress from endosomes, the minor capsid protein L2 accompanies the viral DNA to the nucleus and subsequently to the subnuclear promyelocytic leukemia protein bodies (P. M. Day et al., Proc. Natl. Acad. Sci. USA 101:14252-14257, 2004), suggesting that this protein may be involved in the intracytoplasmic transport of the viral genome. We now demonstrate that the L2 protein is able to interact with the microtubule network via the motor protein dynein. L2 protein was found attached to microtubules after uncoating of incoming human papillomavirus pseudovirions. Based on immunofluorescence and coimmunoprecipitation analyses, the L2 region interacting with dynein is mapped to the C-terminal 40 amino acids. Mutations within this region abrogating the L2/dynein interaction strongly reduce the infectivity of pseudoviruses, indicating that this interaction mediates the minus-end-directed transport of the viral genome along microtubules towards the nucleus.

Assembly and Translocation of Papillomavirus Capsid Proteins

ABSTRACT The major and minor capsid proteins of polyomavirus are preassembled in the cytoplasm and translocated to the nucleus only as a VP1-VP2/VP3 complex. In this study, we describe independent nuclear translocation of the L1 major protein and the L2 minor capsid protein of human papillomavirus type 33 by several approaches. First, we observed that expression and nuclear translocation of L2 in natural lesions precede expression of L1. Second, using a cell culture system for coexpression, we found that accumulation of L2 in nuclear domain 10 (ND10) subnuclear structures precedes L1 by several hours. In contrast, complexes of L2 and mutants of L1 forced to assemble in the cytoplasm are translocated directly to ND10, like L2 expressed alone. Interestingly, accumulation of wild-type L1 is observed only after L2-induced release of the ND10-associated protein Sp100. Third, nuclear translocation of L2 but not of L1 was blocked by the proteasome inhibitor MG132. Our data suggest that L1 and L2 interaction occurs after L2-induced reorganization of ND10 subnuclear domains.

Tetraspanin CD151 Mediates Papillomavirus Type 16 Endocytosis

ABSTRACT Human papillomavirus type 16 (HPV16) is the primary etiologic agent for cervical cancer. The infectious entry of HPV16 into cells occurs via a so-far poorly characterized clathrin- and caveolin-independent endocytic pathway, which involves tetraspanin proteins and actin. In this study, we investigated the specific role of the tetraspanin CD151 in the early steps of HPV16 infection. We show that surface-bound HPV16 moves together with CD151 within the plane of the membrane before they cointernalize into endosomes. Depletion of endogenous CD151 did not affect binding of viral particles to cells but resulted in reduction of HPV16 endocytosis. HPV16 uptake is dependent on the C-terminal cytoplasmic region of CD151 but does not require its tyrosine-based sorting motif. Reexpression of the wild-type CD151 but not mutants affecting integrin functions restored virus internalization in CD151-depleted cells. Accordingly, short interfering RNA (siRNA) gene knockdown experiments confirmed that CD151-associated integrins (i.e., α3β1 and α6β1/4) are involved in HPV16 infection. Furthermore, palmitoylation-deficient CD151 did not support HPV16 cell entry. These data show that complex formation of CD151 with laminin-binding integrins and integration of the complex into tetraspanin-enriched microdomains are critical for HPV16 endocytosis.

Identification of the dynein light chains required for human papillomavirus infection

Human papillomaviruses (HPVs) are a family of small non‐enveloped DNA viruses. Some genital HPV types, including HPV type 16 (HPV16), are the causative agent for the development of cancer at the site of infection. HPVs encode two capsid proteins, L1 and L2. After endocytic cell entry and egress from endosomes, L2 accompanies the viral DNA to the nucleus where replication is initiated. For cytoplasmic transport, L2 interacts with the microtubule network via the motor protein complex dynein. We have performed yeast two‐hybrid screening and identified the dynein light chain DYNLT1 (previously called Tctex1) as interaction partner of HPV16 L2. Using co‐immunoprecipitation and immunofluorescence colocalization studies we confirmed the L2–DYNLT1 interaction in mammalian cells. Further studies revealed that DYNLT3, the second member of the Tctex‐light chain family, also interacts with L2 in vitro and in vivo, whereas other constituents of the dynein complex were not found to associate with L2. Depletion of DYNLT1 and DYNLT3 by specific siRNAs or cytosolic delivery of light chain‐specific antibodies inhibited infection of HPV16. Therefore, this work identified two host cell proteins involved in HPV16 infection that are most likely required for transport purposes towards the nucleus.

Nuclear Translocation of Papillomavirus Minor Capsid Protein L2 Requires Hsc70

ABSTRACT Minor capsid protein L2 of papillomaviruses plays an essential role in virus assembly by recruiting viral components to PML bodies, the proposed sites of virus morphogenesis. We demonstrate here that the function of L2 in virus assembly requires the chaperone Hsc70. Hsc70 was found dispersed in naturally infected keratinocytes and cultured cells. A dramatic relocation of Hsc70 from the cytoplasm to PML bodies was induced in these cells by L2 expression. Hsc70-L2 complex formation was confirmed by coimmunoprecipitation. The complex was modulated by the cochaperones Hip and Bag-1, which stabilize and destabilize Hsc70-substrate complexes, respectively. Cytoplasmic depletion of Hsc70 caused retention of wild-type and N-terminally truncated L2, but not of C-terminally truncated L2, in the cytoplasm. This retention was partially reversed by overexpression of Hsc70 fused to green fluorescent protein but not by ATPase-negative Hsc70. Hsc70 associated with L1-L2 virus-like particles (VLPs) but not with VLPs composed either of L1 alone or of L1 and C-terminally truncated L2. Moreover, displacement of Hsc70 from L1-L2 VLPs by encapsidation of DNA, generating pseudovirions, was found. These data indicate that Hsc70 transiently associates with viral capsids during the integration of L2, possibly via the L2 C terminus. Completion of virus assembly results in displacement of Hsc70 from virions.

Human Papillomavirus Types 16, 18, and 31 Share Similar Endocytic Requirements for Entry

ABSTRACT 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.