Human Papillomavirus 16 L2 Recruits both Retromer and Retriever Complexes during Retrograde Trafficking of the Viral Genome to the Cell Nucleus.

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.

Human Papillomavirus infection requires the CCT Chaperonin Complex.

Human papillomavirus (HPV) infection is a multi-step process that implies complex interactions of the viral particles with cellular proteins. The HPV capsid includes the two structural proteins L1 and L2, that play crucial roles on infectious viral entry. L2 is particularly relevant for the intracellular trafficking of the viral DNA towards 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 analysed 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 (PsVs) infection were revealed by siRNA-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 co-localised 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 PsVs intracellular normal processing when ablating CCT3. Taken together, these studies demonstrate the importance of CCT chaperonin during HPV infectious entry.ImportanceSeveral 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.

Phosphorylation of Human Papillomavirus Type 16 L2 Contributes to Efficient Virus Infectious Entry.

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.

Oncogenic comparison of human papillomavirus type 58 E7 variants.

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony-forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage-independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7-driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow-up strategy.

Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

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.

Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways.

A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells.

Characterizing the spatio-temporal role of sorting nexin 17 in human papillomavirus trafficking.

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.

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation.

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.

A Novel PDZ Domain Interaction Mediates the Binding between Human Papillomavirus 16 L2 and Sorting Nexin 27 and Modulates Virion Trafficking.

UNLABELLED: 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.

Geographical distribution and oncogenic risk association of human papillomavirus type 58 E6 and E7 sequence variations.

Human papillomavirus (HPV) 58 accounts for a notable proportion of cervical cancers in East Asia and parts of Latin America, but it is uncommon elsewhere. The reason for such ethnogeographical predilection is unknown. In our study, nucleotide sequences of E6 and E7 genes of 401 HPV58 isolates collected from 15 countries/cities across four continents were examined. Phylogenetic relationship, geographical distribution and risk association of nucleotide sequence variations were analyzed. We found that the E6 genes of HPV58 variants were more conserved than E7. Thus, E6 is a more appropriate target for type-specific detection, whereas E7 is more appropriate for strain differentiation. The frequency of sequence variation varied geographically. Africa had significantly more isolates with E6-367A (D86E) but significantly less isolates with E6-203G, -245G, -367C (prototype-like) than other regions (p ≤ 0.003). E7-632T, -760A (T20I, G63S) was more frequently found in Asia, and E7-793G (T74A) was more frequent in Africa (p < 0.001). Variants with T20I and G63S substitutions at E7 conferred a significantly higher risk for cervical intraepithelial neoplasia grade III and invasive cervical cancer compared to other HPV58 variants (odds ratio = 4.44, p = 0.007). In conclusion, T20I and/or G63S substitution(s) at E7 of HPV58 is/are associated with a higher risk for cervical neoplasia. These substitutions are more commonly found in Asia and the Americas, which may account for the higher disease attribution of HPV58 in these areas.

Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD.

Human papillomavirus (HPV) E6 oncoproteins target many cellular proteins for ubiquitin-mediated proteasomal degradation. In the case of p53, this is mediated principally by the E6AP ubiquitin ligase. Several studies have reported that E6 can target certain of its substrates in an apparently E6AP-independent fashion and that several of these substrates vary in the degree to which they are degraded by E6 at different stages of malignancy. To more fully understand the regulation of the E6AP/E6 proteolytic targeting complex, we performed a mass spectroscopic analysis of HPV type 18 (HPV-18) E6 protein complexes and identified the HECT domain-containing ubiquitin ligase EDD as a new HPV-18 E6 binding partner. We show that EDD can interact independently with both E6 and E6AP. Furthermore, EDD appears to regulate E6AP expression levels independently of E6, and loss of EDD stimulates the proteolytic activity of the E6/E6AP complex. Conversely, higher levels of EDD expression protect a number of substrates from E6-induced degradation, partly as a consequence of lower levels of E6 and E6AP expression. Intriguingly, reduction in EDD expression levels in HPV-18-positive HeLa cells enhances cell resistance to apoptotic and growth arrest stimuli. These studies suggest that changes in the levels of EDD expression during different stages of the viral life cycle or during malignancy could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV-induced malignancy.

Identification of human papillomavirus type 58 lineages and the distribution worldwide.

BACKGROUND: Human papillomavirus type 58 (HPV-58) accounts for a much higher proportion of cervical cancers in East Asia than other types. A classification system of HPV-58, which is essential for molecular epidemiological study, is lacking. METHODS AND RESULTS: This study analyzed the sequences of 401 isolates collected from 15 countries and cities. The 268 unique concatenated E6-E7-E2-E5-L1-LCR sequences that comprised 57% of the whole HPV-58 genome showed 4 distinct clusters. L1 and LCR produced tree topologies that best resembled the concatenated sequences and thus are the most appropriate surrogate regions for lineage classification. Moreover, short fragments from L1 (nucleotides 6014-6539) and LCR (nucleotides 7257-7429 and 7540-52) were found to contain sequence signatures informative for lineage identification. Lineage A was the most prevalent lineage across all regions. Lineage C was more frequent in Africa than elsewhere, whereas lineage D was more prevalent in Africa than in Asia. Among lineage A variants, sublineage A2 dominated in Africa, the Americas, and Europe, but not in Asia. Sublineage A1, which represents the prototype that originated from a patient with cancer, was rare worldwide except in Asia. CONCLUSIONS: HPV-58 can be classified into 4 lineages that show some degree of ethnogeographic predilection in distribution. The evolutionary, epidemiological, and pathological characteristics of these lineages warrant further study.

HPV-16 E7 Interacts with the Endocytic Machinery via the AP2 Adaptor µ2 Subunit.

Human papillomavirus (HPV) E7 plays a major role in HPV-induced malignancy, perturbing cell cycle regulation, and driving cell proliferation. Major targets of cancer-causing HPV E7 proteins are the pRB family of tumor suppressors, which E7 targets for proteasome-mediated degradation and whose interaction is promoted through an acidic patch, downstream of the LXCXE motif in E7, that is subject to phosphorylation by casein kinase II (CKII). In this study we show that HPV-16 E7 targets the AP2-complex, which plays a critical role in cargo recognition in clathrin-mediated endocytosis. Intriguingly, HPV-16 E7 contains a specific amino acid sequence for AP2 recognition, and this overlaps the pRb LXCXE recognition sequence but involves completely different amino acid residues. HPV-16 E7 does this by binding to the AP2-µ2 adaptor protein subunit via residues 25-YEQL-28 within the LXCXE motif. Point mutations at Y25 within 22-LYCYE-26 suggest that the interaction of E7 with AP2-µ2 is independent from pRB binding. In cells, this interaction is modulated by acidic residues downstream of LXCXE, with the binding being facilitated by CKII-phosphorylation of the serines at positions 31 and 32. Finally, we also show that association of HPV-16 E7 with the AP2 adaptor complex can contribute to cellular transformation under low-nutrient conditions, which appears to be mediated, in part, through inhibition of AP2-mediated internalization of epidermal growth factor receptor (EGFR). This indicates that E7 can modulate endocytic transport pathways, with one such component, EGFR, most likely contributing toward the ability of E7 to induce cell transformation and malignancy. These studies define a new and unexpected role for HPV-16 E7 in targeting clathrin-mediated endocytosis. IMPORTANCE Despite being a very small protein, HPV-E7 has a wide range of functions within the infected cell, many of which can lead to cell transformation. High-risk HPV-E7 deregulates the function of many cellular proteins, perturbing cellular homeostasis. We show that a novel target of HPV-E7 is the clathrin-adaptor protein 2 complex (AP2) µ2 subunit, interacting via residues within E7's pRB-binding region. Mutational studies show that an AP2 recognition motif is present in the CR2 region and is conserved in >50 HPV types, suggesting a common function for this motif in HPV biology. Mutational analysis suggests that this motif is important for cellular transformation, potentially modulating endocytosis of growth factor receptors such as EGFR, and thus being a novel activity of E7 in modulating clathrin-mediated endocytosis and cargo selection. This study has important implications for the molecular basis of E7 function in modulating protein trafficking at the cell surface.

Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells.

BACKGROUND: "High risk" human papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins. METHODS: In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™ technology and Surface Plasmon Resonance. RESULTS: The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner. CONCLUSIONS: Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the HPV-associated lesions.

The human papillomavirus (HPV) E6* proteins from high-risk, mucosal HPVs can direct degradation of cellular proteins in the absence of full-length E6 protein.

The E6 oncoproteins from high-risk mucosotrophic human papillomaviruses (HPVs) target a range of cellular proteins for proteasome-mediated degradation. Apart from the tumor suppressor p53 and proapoptotic Bcl-2 family member Bak, many targets contain class 1 PDZ domains and are involved in cell junction stability and signaling. The targeting mechanism is considered to function by the E6 protein acting as an adaptor molecule linking a cellular ubiquitin ligase to the target protein. In each case, whether the target is the p53 tumor suppressor or a member of the group of PDZ domain-containing targets, this mechanism relies on a direct interaction between E6 and its cellular target. This study focuses on the impact of the HPV type 18 (HPV-18) E6*I protein on the stability of Akt, Dlg, MAGI-1, MAGI-2, and Scribble. We show that HPV-18 E6* expression can downregulate the expression levels of Akt, Dlg, and Scribble in the absence of full-length HPV-18 E6 protein. The reduction in Dlg levels by E6* is independent of transcription and does not require a direct interaction between the two proteins although the proteasome pathway is involved. Further, we provide evidence that activation of certain signal transduction pathways has a profound effect on the targeting of Dlg by E6* and suggest that high-risk HPV E6 oncoproteins can target certain substrates both directly and indirectly through the E6* proteins and may cooperate in their degradation.

Viruses and the 26S proteasome: hacking into destruction.

The discovery that the human papillomavirus E6 oncoprotein could direct the ubiquitination and degradation of the p53 tumour suppressor at the 26S proteasome was the beginning of a new view on virus-host interactions. A decade later, a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. These activities are required for various aspects of the virus life-cycle from entry, through replication and enhanced cell survival, to viral release. As with oncogenes and cell-cycle control, the study of apparently simple viruses has provided a wealth of information on the function of a whole class of cellular proteins whose function is arguably as important as that of the kinases: the ubiquitin-protein ligases.

Choosing the right path: membrane trafficking and infectious entry of small DNA tumor viruses.

To infect mammalian cells, all infectious viruses must cross a common set of biophysical membrane barriers to gain access to the cell. The virus capsid proteins attach to a host cell, become endocytosed, and traffic the viral genome to sites of replication. To do this they must interact with the membrane-confined organelles that control endocytosis, endosomal sorting, processing, and degradation of biological molecules. In this review, we highlight some recent advances in our understanding of the mechanisms that small non-enveloped DNA tumor viruses, such as Human Papillomavirus (HPV) and Polyomaviruses (PyV) employ to attain infectious entry. These viruses exploit different pathways to mediate entry, uncoating and subsequent transport to the nucleus via the Trans Golgi Network (TGN) or the Endoplasmic Reticulum (ER). Understanding how the viral capsid proteins interact with cellular membranous organelles sheds light on the novel ways by which viruses can hi-jack endocytic transport pathways and provides unique insights into how the highly complex machinery controlling cargo fate determination is regulated within the cell.

Diverse Papillomavirus Types Induce Endosomal Tubulation.

Previous studies have shown that the endoplasmic reticulum (ER)-anchored protein VAP is strictly required by human papillomavirus type 16 (HPV-16) for successful infectious entry. Entry appeared to be mediated in part through the induction of endosomal tubulation and subsequent transport of the virion to the trans-Golgi network (TGN). In this study, we were interested in investigating whether this mechanism of infectious entry is conserved across multiple Papillomavirus types. To do this, we analyzed the role of VAP and endosomal tubulation following infection with Pseudovirions (PsVs) derived from the alpha, beta, delta, kappa, and pi papillomavirus genera, reflecting viruses that are important human and animal pathogens. We demonstrate that VAP is essential for infection with all PV types analyzed. Furthermore, we find that VAP and EGFR-dependent endosomal tubulation is also induced by all these different Papillomaviruses. These results indicate an evolutionarily conserved requirement for VAP-induced endocytic tubulation during Papillomavirus infectious entry.

The VPS4 component of the ESCRT machinery plays an essential role in HPV infectious entry and capsid disassembly.

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.

Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling.

Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5ß1 integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.