Retromer-Dependent Cell Entry by Human Papillomaviruses

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.