The Human Papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection, but the molecular mechanisms underlying its mode of action remain obscure. Using a proteomic approach we have identified the adaptor protein, Sorting Nexin 17 (SNX17) as a strong interacting partner of HPV L2. This interaction occurs through a highly conserved SNX17 consensus binding motif, which is present in the majority of HPV L2 proteins analysed. Using mutants of L2 defective for SNX17 interaction, or siRNA ablation of SNX17 expression we demonstrate that the interaction between L2 and SNX17 is essential for viral infection. Furthermore, loss of the L2-SNX17 interaction results in enhanced turnover of the L2 protein and decreased stability of the viral capsids, and concomitantly there is a dramatic decrease in the efficiency with which viral genomes transit to the nucleus. Indeed, using a range of endosomal and lysosomal markers we show that capsids defective in their capacity to bind SNX17 transit much more rapidly to the lysosomal compartment. These results demonstrate that the L2-SNX17 interaction is essential for viral infection and facilitates the escape of the L2-DNA complex from the late endosomal/lysosomal compartments.

This work focuses on the photocatalytic performances and antibacterial activity of TiO 2 and Au/TiO 2 nanosystems. While the former are obtained by a sol-gel route, the latter are synthesized by an innovative hybrid RF-sputtering/sol-gel approach, followed by ex situ annealing in air up to 600 • C. Important information on nanoparticle size, shape and distribution is obtained by the combined use of glancing incidence x-ray diffraction (GIXRD) and field emission-scanning electron microscopy (FE-SEM). Subsequently, the photocatalytic performances of the obtained nanosystems in the decomposition of the azo-dye Plasmocorinth B and their antibacterial activity in the elimination of Bacillus subtilis are illustrated and discussed in comparison with films obtained from standard Degussa P25 powders. The obtained results show a significant dependence of the functional performances on the system's compositional, structural and morphological properties. In particular, the dispersion of gold nanoparticles on the TiO 2 matrix has a beneficial influence on the azo-dye photodegradation, whereas the antimicrobial activity of Au/TiO 2 films is retarded with respect to pure TiO 2 .

The human papillomavirus (HPV) E6 oncoprotein is fundamental to the ability of these viruses to induce human malignancy. A defining characteristic of the HPV E6 oncoproteins found in cancer‐causing HPV types is the presence of a PDZ binding motif at their extreme C‐terminus. Through this motif, E6 is able to interact with a large number of cellular proteins that contain PDZ domains. Many of these cellular proteins are involved in regulation of processes associated with the control of cell attachment, cell proliferation, cell polarity and cell signaling. How E6 targets multiple proteins containing the same recognition domain is still an open question. In this review, we highlight aspects of E6 function and biology that help to answer this question, and thereby provide insight into the role of these substrates during development of HPV‐induced malignancy.

Previous studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-FERM family member sorting nexin 27 (SNX27), which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct and, unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that small interfering RNA (siRNA)-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection but that double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for the knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery. IMPORTANCEThe trafficking of papillomaviruses to the host cell nucleus during their natural infectious life cycle is an incompletely understood process. Studies have suggested that the virus minor capsid protein L2 can interact with the endosomal recycling pathway, in part by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-Golgi network rather than back to the plasma membrane. In this study, we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics and provides potential tools for the further dissection of endosomal structure and function.T he entry of papillomaviruses (PVs) into host cells involves a series of coordinated steps whereby virus particles attach to cell surface receptors and are internalized into endocytic vesicles. This allows the virus capsid proteins, tethered to the viral genome, to interact with cellular protein complexes within endosomes, which are exploited to allow virion disassembly, followed by the transport of L2 and the viral genome to the nucleus and their entry into the nucleus, for subsequent expression of viral transcripts. The precise details of the first step in this cascade of events remain, to a degree, controversial, although it seems that different papillomavirus types exploit different mechanisms to gain entry to the cell (1-3). Subsequent steps during viral infection may be common to all papillomavirus types and, although incompletely understood, involve virion disassembly during endosome acidification and subsequent separation of L1 and L2. While the majority of L1 is sorted into lysosomes, L2, tethered to the viral genome, is transported through the trans-Golgi network (4) and finally enters the nucleus during mitosis (5), when the nuclear membrane is dissolved, where it loca...

Human papillomavirus (HPV) infection is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells are controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase, and subsequent transfer of the virus to an as-yet-unidentified high-affinity receptor. In line with this model, we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were nonpermissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pulldown assays followed by mass spectrometry analysis showed that furin-precleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and uncleaved HPV16-PsVs colocalized with surface-expressed vimentin on pgsD677, HeLa, HaCaT, and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by small interfering RNA (siRNA) knockdown in HeLa and NIKS cells significantly increased HPV16-PsV infectious internalization, while overexpression of vimentin had the opposite effect. The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for the design of novel antiviral drugs preventing HPV internalization into epithelial cells.IMPORTANCE Despite HPV being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalization are poorly understood. We herein describe the identification of surface-expressed vimentin as a novel molecule not previously implicated in the infectious internalization of HPV16. Contrary to our expectations, vimentin was found to act not as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalization of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.

Previous studies have shown that the human papillomavirus type 16 (HPV-16) L2 capsid protein plays an essential role in viral infection, in part through its interaction with sorting nexin 17 (SNX17). We now show that this interaction between L2 and SNX17 is conserved across multiple PV types. Furthermore, we demonstrate that SNX17 is essential for infection with all PV types analyzed, indicating an evolutionarily highly conserved virus entry mechanism. Many of the more than 120 known human papillomaviruses (HPVs) cause sexually transmitted infections, with a subset of them being associated with the development of epithelial tumors and cancer (1). Infectious entry of HPVs is still poorly characterized in terms of the cellular proteins and endocytic pathways employed. It seems that some HPV types may have evolved different entry strategies (2, 3) or take advantage of cross talk between different routes of endocytosis (4,5). A recent study suggested that HPVs can usurp soluble cellular factors for interaction with a plethora of entry receptors, resulting in different routes of internalization (6). The HPV capsid, comprising the major L1 and minor L2 proteins, fulfills various roles in the establishment of a successful viral infection. L2 is critically involved in many steps of the infection process, although its precise functions in entry, intracellular trafficking, endosomal escape, and nuclear import of the viral genome have not been fully elucidated (7). While L2 is not essential for viral uncoating, it is critical for the escape of viral DNA from the endosomal compartment (8) and ultimately for aiding transport of the viral genome to the nucleus (9-11).We reported previously that the HPV-16 L2 interaction with a member of the sorting nexin family, SNX17, is essential for HPV-16 infection (12). SNX17 is localized in early endosomes and recycling tubules and is primarily involved in endosomal recycling (13,14). We showed that at least one putative SNX17 binding motif, NPxF/Y (PTB consensus binding motif) is present in L2s from diverse papillomavirus (PV) genera, indicating that this motif is evolutionarily highly conserved. A previous protein mass spectrometry analysis of HPV-11 L2 and HPV-16 L2 immunoprecipitates in HEK293 cells revealed that SNX17 is an interacting partner of both L2 proteins (12). To investigate whether SNX17 interacts with L2 proteins from diverse HPV genera, a series of pulldown assays with a panel of glutathione S-transferase (GST)-L2 proteins and endogenously derived SNX17 were performed. The results (Fig. 1A) confirmed the interaction between HPV-16 and HPV-11 L2 (genus Alpha) proteins and SNX17. The analysis was then extended to HPV-5 L2 (genus Beta), bovine papillomavirus type 1 (BPV-1) L2 (genus Delta), and cottontail rabbit papillomavirus type 1 (SfPV-1) L2 (genus Kappa). The results in Fig. 1A show interactions between these diverse L2 proteins and SNX17, although the interaction between SNX17 and SfPV L2 was consistently weaker.Previous analyses indicated that HPV-16 L2 bound SNX17 ...

The human papillomavirus (HPV) minor capsid protein L2 plays important roles in the generation of infectious viral particles and in the initial steps of infection. Here we show that HPV-16 L2 protein is sumoylated at lysine 35 and that sumoylation affects its stability. Interestingly, the sumoylated form of L2 cannot bind to the major capsid protein L1, suggesting a mechanism by which capsid assembly may be modulated in an infected cell. Additionally, L2 appears to modulate the overall sumoylation status of the host cell. These observations indicate a complex interplay between the HPV L2 protein and the host sumoylation machinery.Posttranslational modification is a central method of diversifying protein function. Ubiquitin-like proteins such as SUMO (small ubiquitin-related modifier) are known to be key regulators of several biological functions (31). In humans, at least three SUMO forms (SUMO1, -2, and -3) are expressed. SUMO2 and SUMO3 (here called SUMO2/3) are closely related, sharing 97% identity, whereas SUMO1 shares 43% identity with SUMO2/3. SUMO modification exerts a variety of effects on its targets, altering a target's cellular localization, its stability, its ability to interact with other proteins, and its activity (31). Most known sumoylation targets are transcription factors or other proteins involved in chromatin structure, regulation, and expression (12), supporting a fundamental role for this modification system in regulating cellular homeostasis. Hence, it is no surprise that viral proteins can exploit the host sumoylation system; proteins from both RNA and DNA viruses have been shown to be sumoylated and/or to interact with the sumoylation machinery (8). The viruses that exhibit interplay between sumoylation and viral proteins can be divided into two groups: viruses that have their proteins sumoylated and viruses whose proteins directly modify host sumoylation. In both cases, the outcome is a cellular environment more favorable for viral replication (5, 8).Human papillomavirus (HPV) infects both mucosal and cutaneous epithelia, and certain high-risk HPV types are the causative agents of cervical cancer (11). Two structural proteins, L1 and L2, form the papillomavirus capsid (16). The minor capsid protein L2 plays a critical role in the generation of infectious viral particles and in early events of HPV infection (13), although its precise functions in HPV entry, intracellular trafficking, endosomal escape, and the nuclear import of the HPV genome have not been fully elucidated (21).Previous studies have shown sumoylation to be important in the HPV life cycle. The functions of two early proteins, E1 and E2, are modified directly by sumoylation (17,19,26,28), and three others, E2 (27), E6 (3), and E7 (14), affect host sumoylation pathways.HPV type 16 (HPV-16) L2's potential involvement with the sumoylation machinery has not been documented, although a consensus sequence, ⌿KXE, where ⌿ is a large hydrophobic amino acid, corresponding to the SUMO acceptor site in most known SUMO substrate proteins...

Infection with human papillomaviruses (HPV) requires the minor capsid component L2, which plays an essential role in directing appropriate endosomal trafficking. Previous studies have indicated an infection route involving multi-vesicular bodies (MVBs), and an essential element in their biogenesis is the ESCRT machinery. Here we show that the ESCRT component TSG101 is required for optimal infection with both HPV-16 and BPV-1, with loss of TSG101 resulting in a decrease in viral infection, whereas overexpressed TSG101 increases rates of infection. We find that L2 proteins from multiple PV types interact with TSG101 and show that this interaction contributes to an alteration in the subcellular distribution of L2. In addition, TSG101 can modulate the levels of L2 polyubiquitination. These results demonstrate that TSG101 plays an important part in infection with diverse PVs, and suggests that trafficking of HPV through the ESCRT machinery and MVBs is part of infectious virus entry.