During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.HPV entry | vesicular transport | mitosis | digitonin | nuclear vesicle A major hurdle of DNA viruses during a primary infection is successful navigation of the cytoplasm to deliver the viral genome to the nucleus. Foreign DNA that enters the cytoplasm is susceptible to being sensed by innate immune sensors (1). Not surprisingly, viruses have evolved mechanisms to evade detection by these sensors. For example, herpesviruses and adenoviruses both egress into the cytoplasm, yet protect their viral DNA by keeping it encased in viral capsids until directly transferring it into the nucleus through the nuclear pore complex. In contrast, the capsid is unlikely to protect papillomavirus (PV) genomes while traversing the cytoplasm, because the major capsid protein is lost in the endocytic compartment (2).The PV capsid is composed of two viral proteins, the major capsid protein L1 and the minor capsid protein L2, which enclose a chromatinized, circular, double-stranded DNA genome ∼8 kb in size (3-6). Following primary attachment and internalization, acidification of early endosomes triggers capsid disassembly (7-22). Host cell cyclophilins release the majority of L1 from the L2 protein, which remains in complex with the viral genome (2,23,24). A large portion of the L2 protein translocates across the endocytic membrane to engage factors, including the retromer complex, dynein, sorting nexins, and rab GTPases, that mediate transport to the trans-Golgi network (TGN) (25)(26)(27)(28)(29)(30)(31)(32)(33)). An siRNA screen has suggested that nuclear pore complexes are not required for nuclear entry, but that nuclear envelope breakdown during mitosis is necessary (34, 35).Currently, when and how the human PV (HPV) genome egresses from the membranous compartment is unclear. Here we present evidence indicating that after the onset of mitosis, the viral genome of HPV type 16 (HPV16), an HPV type...
Viruses employ elaborate strategies to coopt the cellular processes they require to replicate while simultaneously thwarting host antiviral responses. In many instances, how this is accomplished remains poorly understood. Here, we identify a protein, F17 encoded by cytoplasmically replicating poxviruses, that binds and sequesters Raptor and Rictor, regulators of mammalian target of rapamycin complexes mTORC1 and mTORC2, respectively. This disrupts mTORC1-mTORC2 crosstalk that coordinates host responses to poxvirus infection. During infection with poxvirus lacking F17, cGAS accumulates together with endoplasmic reticulum vesicles around the Golgi, where activated STING puncta form, leading to interferon-stimulated gene expression. By contrast, poxvirus expressing F17 dysregulates mTOR, which localizes to the Golgi and blocks these antiviral responses in part through mTOR-dependent cGAS degradation. Ancestral conservation of Raptor/Rictor across eukaryotes, along with expression of F17 across poxviruses, suggests that mTOR dysregulation forms a conserved poxvirus strategy to counter cytosolic sensing while maintaining the metabolic benefits of mTOR activity.
The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. IMPORTANCEIn order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors. Human papillomaviruses (HPVs) are nonenveloped DNA tumor viruses that infect the skin and mucosal epithelial cells. Infection by HPVs can induce hyper-proliferative lesions of the tissues, whereupon high-risk HPVs can progress to malignant tumors. Of the high-risk HPVs, 70% of cervical cancer can be attributed to HPVs 16 and 18 (1). These HPVs are associated with a variety of anogenital and oral carcinomas. Despite the success of the prophylactic vaccine against these high-risk HPVs, HPV-associated cancers will continue to be a global health burden in the future due to low vaccination rates and lack of therapeutic effect (2). Therefore, uncovering the conserved steps of how HPVs establish infection can be used to find antiviral targets that may be less virus restrictive than the current vaccines.The HPV capsid is composed of 360 molecules of the major capsid protein, L1, and up to 72 molecules of the minor capsid protein, L2 (3-6). Much of our advancement in understanding HPV entry can be attributed to the efficient generation of pseudovirions. With respect to cellular entry, pseudovirions are considered to be indistinguishable from native virions but encapsidate a reporter plasmid (pseudogenome) in place of the HP...
The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels.
During infectious entry, acidification within the endosome triggers uncoating of the human papillomavirus (HPV) capsid, whereupon host cyclophilins facilitate the release of most of the major capsid protein, L1, from the minor capsid protein L2 and the viral genome. The L2/DNA complex traffics to the trans-Golgi network (TGN). After the onset of mitosis, HPV-harboring transport vesicles bud from the TGN, followed by association with mitotic chromosomes. During this time, the HPV genome remains in a vesicular compartment until the nucleus has completely reformed. Recent data suggest that while most of L1 protein dissociates and is degraded in the endosome, some L1 protein remains associated with the viral genome. The L1 protein has DNA binding activity, and the L2 protein has multiple domains capable of interacting with L1 capsomeres. In this study, we report that some L1 protein traffics with L2 and viral genome to the nucleus. The accompanying L1 protein is mostly full length and retains conformation-dependent epitopes, which are recognized by neutralizing antibodies. Since more than one L1 molecule contributes to these epitopes and requires assembly into capsomeres, we propose that L1 protein is present in the form of pentamers. Furthermore, we provide evidence that the L1 protein interacts directly with viral DNA within the capsid. Based on our findings, we propose that the L1 protein, likely arranged as capsomeres, stabilizes the viral genome within the subviral complex during intracellular trafficking.IMPORTANCE After internalization, the nonenveloped human papillomavirus virion uncoats in the endosome, whereupon conformational changes result in a dissociation of a subset of the major capsid protein L1 from the minor capsid protein L2, which remains in complex with the viral DNA. Recent data suggest that some L1 protein may accompany the viral genome beyond the endosomal compartment. We demonstrate that conformationally intact L1 protein, likely still arranged as capsomeres, remains associated with the incoming viral genome throughout mitosis and transiently resides in the nucleus until after the viral DNA is released from the transport vesicle.KEYWORDS HPV entry, HPV16, L1 protein, L2 protein, mitosis, nuclear transport, virus trafficking P apillomaviruses are a family of nonenveloped DNA viruses that infect a wide range of hosts with a preference for epithelial cells. The papillomaviruses that infect humans (HPVs) are a particular health burden. While most infections with HPVs are cleared by the immune system, a persistent infection with the high-risk types is associated with increased risk of carcinomas. The high-risk type 16 accounts for
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