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Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2 cycl , an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2 cycl inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2 cycl likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2 cycl as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking. IMPORTANCE The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2 cycl protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2 cycl treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2 cycl may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts. The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2 cycl protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2 cycl treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2 cycl may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts.

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.

Simian virus 40 (SV40) is a nonenveloped DNA virus that traffics through the endoplasmic reticulum (ER) en route to the nucleus, but the mechanisms of capsid disassembly and ER exit are poorly understood. We conducted an unbiased RNA interference screen to identify cellular genes required for SV40 infection. SV40 infection was specifically inhibited by up to 50-fold by knockdown of four different DNAJ molecular cochaperones or by inhibition of BiP, the Hsp70 partner of DNAJB11. These proteins were not required for the initiation of capsid disassembly, but knockdown markedly inhibited SV40 exit from the ER. In addition, BiP formed a complex with SV40 capsids in the ER in a DNAJB11-dependent fashion. These experiments identify five new cellular proteins required for SV40 infection and suggest that the binding of BiP to the capsid is required for ER exit. Further studies of these proteins will provide insight into the molecular mechanisms of polyomavirus infection and ER function. IMPORTANCE The polyomaviruses, including simian virus 40 (SV40), are important human pathogens and model systems for exploring the general features of virus replication and cell biology. We used a genetic system to interrogate the role of cellular genes in SV40 infection. Based on the results of this unbiased genetic screen and analysis of proteins related to the strongest hit from the screen, we identified five new cellular proteins required for the entry of SV40 into cells. These proteins physically associate with SV40 in the endoplasmic reticulum (ER) during virus entry and are required for exit of the partially disassembled virus from this organelle. These results demonstrate that the polyomaviruses have coopted an ER-localized protein quality control process to initiate disassembly and transit through the cell on their way to the nuclear site of virus replication. The polyomaviruses, including simian virus 40 (SV40), are important human pathogens and model systems for exploring the general features of virus replication and cell biology. We used a genetic system to interrogate the role of cellular genes in SV40 infection. Based on the results of this unbiased genetic screen and analysis of proteins related to the strongest hit from the screen, we identified five new cellular proteins required for the entry of SV40 into cells. These proteins physically associate with SV40 in the endoplasmic reticulum (ER) during virus entry and are required for exit of the partially disassembled virus from this organelle. These results demonstrate that the polyomaviruses have coopted an ER-localized protein quality control process to initiate disassembly and transit through the cell on their way to the nuclear site of virus replication.

We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

Human papillomaviruses (HPVs) are widespread human pathogens that are responsible for 5% of all human cancers. Despite significant advances in our understanding of the HPV life cycle, the mechanisms responsible for HPV cell entry have remained very poorly understood. In particular, the identity of the cellular factors and pathways mediating viral endocytosis and trafficking is a subject of much controversy. Recent improvements in systems for virus production have allowed the efficient production of large quantities of HPV pseudovirions (PsVs) that can be used to study the process of infection. In this work we used a genome-wide RNA interference screen to uncover all the human genes required for infection by HPV type 16 (HPV16). HPV16 is a \"high-risk\" virus that is linked to 50% of all cervical cancer cases, and a significant portion of anogenital and oropharyngeal malignancies. The screen identified a large number of cellular genes that have not been previously implicated in any HPV infection. Importantly, the screen uncovered a role for the Golgi apparatus and the retrograde transport pathway in viral cell entry. HPV16 was observed to localize to the trans-Golgi network (TGN) and the Golgi late after infection. Chemical or genetic inhibition of early endosome-to-TGN transport decreased viral infection efficiency and reduced HPV16 co-localization with the Golgi. Viral trafficking to the Golgi was found to require the activity of the retromer, an evolutionary conserved protein complex that functions as a sorting device for the recycling of endosomal proteins to the TGN. The retromer physically associates with incoming HPV16 capsids and initiates their sorting into vesicular carriers that shuttle to the TGN. The retromer was required for infection by all HPV types and in all cell lines tested to date, possibly indicating its essential role in infection by many different HPVs. Notably, a role for the retromer has not been reported in cell entry by any virus. Taken together, results of the genome-wide screen have implicated a number of novel cellular factors, pathways, and organelles in HPV infection. Findings from the screen also provide a starting point for the investigation of additional aspects of infection, such as the nature of the endocytic receptor and mode of endocytosis. Finally, the observation of viral trafficking to the Golgi apparatus challenges current assumptions of HPV16 transfer into the cytoplasm and suggests that the virus escapes the endosomal compartment via retromer sorting into membrane-bound transport intermediates.