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Human papillomavirus (HPV)-induced carcinogenesis is associated with unregulated expression of the oncoproteins E6 and E7. HPV E7 is a viral protein that lacks enzymatic activity; however, it can target several cellular proteins to induce cell transformation and promote uncontrolled proliferation. Although several E7 targets have been described, there are still gaps in the understanding of how this oncoprotein drives cells toward malignancy. Here, using a small HPV type 16 (HPV16) E7 peptide in a proteomic approach, we report Memo1 as a new E7 binding partner, interacting through the aspartic and glutamic acid residues (E80 and D81) in the C-terminal region of HPV16 E7. Furthermore, we demonstrate that HPV16 E7 targets Memo1 for proteasomal degradation through a Cullin2-dependent mechanism. In addition, we show that overexpression of Memo1 decreases cell transformation and proliferation and that reduction of Memo1 levels correlate with activation of Akt and an increase in invasion of HPV-positive cervical cancer cell lines. Our results show a novel HPV E7 interacting partner and describe novel functions of Memo1 in the context of HPV-induced malignancy. IMPORTANCE Although numerous targets have been reported to interact with the HPV E7 oncoprotein, the mechanisms involved in HPV-induced carcinogenesis and the maintenance of cell transformation are still lacking. Here, through pulldown assays using a peptide encompassing the C-terminal region of HPV16 E7, we report Memo1 as a novel E7 interactor. High levels of Memo1 correlated with reduced cell proliferation and, concordantly, knockdown of Memo1 resulted in Akt activation in HPV-positive cell lines. These results highlight new mechanisms used by HPV oncoproteins to modulate proliferation pathways in cervical cancer cells and increase our understanding of the link between Memo1 protein and cancer.

Human papillomavirus (HPV) infection is a multistep process that implies complex interactions of the viral particles with cellular proteins. The HPV capsid includes the two structural proteins L1 and L2, which play crucial roles in infectious viral entry. L2 is particularly relevant for the intracellular trafficking of the viral DNA toward the nucleus. Here, using proteomic studies we identified CCT proteins as novel interaction partners of HPV-16 L2. The CCT multimeric complex is an essential chaperonin which interacts with a large number of protein targets. We analyzed the binding of different components of the CCT complex to L2. We confirmed the interaction of this structural viral protein with the CCT subunit 3 (CCT3), and we found that this interaction requires the N-terminal region of L2. Defects in HPV-16 pseudoviral particle (PsV) infection were revealed by small interfering RNA-mediated knockdown of some CCT subunits. While a substantial drop in the viral infection was associated with the ablation of CCT component 2, even more pronounced effects on infectivity were observed upon depletion of CCT component 3. Using confocal immunofluorescence assays, CCT3 colocalized with HPV PsVs at early times after infection, with L2 being required for this to occur. Further analysis showed the colocalization of several other subunits of CCT with the PsVs. Moreover, we observed a defect in capsid uncoating and a change in PsV intracellular normal processing when ablating CCT3. Taken together, these studies demonstrate the importance of CCT chaperonin during HPV infectious entry. IMPORTANCE Several of the mechanisms that function during the infection of target cells by HPV particles have been previously described. However, many aspects of this process remain unknown. In particular, the role of cellular proteins functioning as molecular chaperones during HPV infections has been only partially investigated. To the best of our knowledge, we describe here for the first time a requirement of the CCT chaperonin for HPV infection. The role of this cellular complex seems to be determined by the binding of its component 3 to the viral structural protein L2. However, CCT’s effect on HPV infection most probably comprises the whole chaperonin complex. Altogether, these studies define an important role for the CCT chaperonin in the processing and intracellular trafficking of HPV particles and in subsequent viral infectious entry.

Previous studies have identified an interaction between the human papillomavirus (HPV) L2 minor capsid protein and sorting nexins 17 and 27 (SNX17 and SNX27) during virus infection. Further studies show the involvement of both retromer and retriever complexes in this process since knockdown of proteins from either complex impairs infection. In this study, we show that HPV L2 and 5-ethynyl-2′-deoxyuridine (EdU)-labeled pseudovirions colocalize with both retromer and retriever, with components of each complex being bound by L2 during infection. We also show that both sorting nexins may interact with either of the recycling complexes but that the interaction between SNX17 and HPV16 L2 is not responsible for retriever recruitment during infection, instead being required for retromer recruitment. Furthermore, we show that retriever recruitment most likely involves a direct interaction between L2 and the C16orf62 subunit of the retriever, in a manner similar to that of its interaction with the VPS35 subunit of retromer. IMPORTANCE Previous studies identified sorting nexins 17 and 27, as well as the retromer complex, as playing a role in HPV infection. This study shows that the newly identified retriever complex also plays an important role and begins to shed light on how both sorting nexins contribute to retromer and retriever recruitment during the infection process.

Wirusy onkogenne (onkowirusy) są zaangażowane w powstawanie około 12% nowotworów u ludzi. Obecnie wirusy, o których wiadomo, że powodują raka, to wirusy zapalenia wątroby typu C i B (HCV i HBV), wirusy brodawczaka ludzkiego (HPV), wirus polyoma komórek Merkla (MCV), ludzki herpeswirus-8 (HHV-8) i ludzki wirus limfotropowy komórek T (HTLV-1). Wirusy nie są jednak pełnymi kancerogenami i w procesie transformacji nowotworowej odgrywają różną rolę. Onkowirusy mogą bezpośrednio zakłócać funkcjonowanie genów kodujących komórkowe białka regulatorowe w wyniku insercji własnego genomu do genomu komórkowego. Mają także własne geny kodujące białka, które zaburzają regulację procesów komórkowych lub zawierają onkogeny wirusowe v-onc, które mogą brać udział bezpośrednio w rozwoju procesu nowotworowego.

The human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection. Previous studies have shown that the interaction between HPV L2 and endosomal sorting nexin 17 (SNX17) is conserved across multiple PV types where it plays an essential role in infectious entry, suggesting an evolutionarily conserved pathway of PV trafficking. Here we show that the peak time of interaction between HPV-16 L2 and SNX17 is rather early, at 2 h post-infection. Interestingly, the L2-SNX17 interaction appears to be important for facilitating capsid disassembly and L1 dissociation, suggesting that L2 recruitment of SNX17 occurs prior to capsid disassembly. Furthermore, we also found evidence of L2-SNX17 association at the later stages of infectious entry, suggesting that the SNX17-mediated sorting machinery is either involved at different stages of HPV trafficking or that L2-SNX17 interaction is a long-lasting event in HPV trafficking.

The human papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are posttranslationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and bovine papillomavirus 1 (BPV-1) L2 proteins. The phospho-modification of L2 and its presence in HPV pseudovirions (PsVs) were confirmed using anti-phospho-L2-specific antibodies. Mutation of the phospho-acceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiencies of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry. IMPORTANCE The papillomavirus L2 capsid protein plays an essential role in infectious entry, where it directs the successful trafficking of incoming viral genomes to the nucleus. However, nothing is known about how potential posttranslational modifications may affect different aspects of capsid assembly or infectious entry. In this study, we report the first phospho-specific modification of the BPV-1 and HPV-16 L2 capsid proteins. The phospho-acceptor site is very highly conserved across multiple papillomavirus types, indicating a highly conserved function within the L2 protein and the viral capsid. We show that this modification plays an essential role in infectious entry, where it modulates susceptibility of the incoming virus to capsid disassembly. These studies therefore define a completely new means of regulating the papillomavirus L2 proteins, a regulation that optimizes endocytic processing and subsequent completion of the infectious entry pathway.

Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of VAP blocks HPV infectious entry at a step after capsid uncoating but prior to localization at the trans -Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry.

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. IMPORTANCE The 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.

Previous studies have indicated that human papillomavirus (HPV) infectious entry is slow, requiring many hours after initial infection for the virus to gain entry into the nucleus. However, intracellular transport pathways typically are very rapid, and in the context of a natural HPV infection in a wounded epithelium, such slow intracellular transport would seem to be at odds with a normal viral infection. Using synchronized cell populations, we show that HPV trafficking can be a rapid process. In cells that are infected in the late S-early G 2 /M phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is detectable by 8 h postinfection. Likewise, reporter DNA can be visualized within the nucleus in conjunction with PML nuclear bodies 1 h to 2 h postinfection in cells that are infected with PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is rapid, with mitosis being the main restriction on nuclear entry. IMPORTANCE HPV infectious entry appears to be slow and requires mitosis to occur before the incoming viral DNA can access the nucleus. In this study, we show that HPV trafficking in the cell actually is very rapid. This demonstrates that in the context of a normal virus infection, the cell cycle state will have a major influence on the time it takes for an incoming virus to enter the nucleus and initiate viral gene expression.

Human Papillomavirus (HPV) infection involves multiple steps, from cell attachment, through endocytic trafficking towards the trans-Golgi network, and, ultimately, the entry into the nucleus during mitosis. An essential viral protein in infectious entry is the minor capsid protein L2, which engages different components of the endocytic sorting machinery during this process. The ESCRT machinery is one such component that seems to play an important role in the early stages of infection. Here we have analysed the role of specific ESCRT components in HPV infection, and we find an essential role for VPS4. Loss of VPS4 blocks infection with multiple PV types, suggesting an evolutionarily conserved critical step in infectious entry. Intriguingly, both L1 and L2 can interact with VPS4, and appear to be in complex with VPS4 during the early stages of virus infection. By using cell lines stably expressing a dominant-negative mutant form of VPS4, we also show that loss of VPS4 ATPase activity results in a marked delay in capsid uncoating, resulting in a defect in the endocytic transport of incoming PsVs. These results demonstrate that the ESCRT machinery, and in particular VPS4, plays a critical role in the early stages of PV infection.

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.

Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.

About 15–20% of human cancers worldwide have viral etiology. Emerging data clearly indicate that several human DNA and RNA viruses, such as human papillomavirus, Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, hepatitis B virus, hepatitis C virus, and human T-cell lymphotropic virus, contribute to cancer development. Human tumor-associated viruses have evolved multiple molecular mechanisms to disrupt specific cellular pathways to facilitate aberrant replication. Although oncogenic viruses belong to different families, their strategies in human cancer development show many similarities and involve viral-encoded oncoproteins targeting the key cellular proteins that regulate cell growth. Recent studies show that virus and host interactions also occur at the epigenetic level. In this review, we summarize the published information related to the interactions between viral proteins and epigenetic machinery which lead to alterations in the epigenetic landscape of the cell contributing to carcinogenesis.