title: Appraising the roles of CBLL1 and the ubiquitin/proteasome system for flavivirus entry and replication.
abstract: The ubiquitin ligase CBLL1 (also known as HAKAI) has been proposed to be a critical cellular factor exploited by West Nile virus (WNV) for productive infection. CBLL1 has emerged as a major hit in a recent RNA interference screen designed to identify cellular factors required for the early stages of the WNV life cycle. Follow-up experiments showed that HeLa cells knocked down for CBLL1 by a small interfering RNA (siRNA) failed to internalize WNV particles and resisted infection. Furthermore, depletion of a free-ubiquitin pool by the proteasome inhibitor MG132 abolished WNV endocytosis, suggesting that CBLL1 acts in concert with the ubiquitin proteasome system to mediate virus internalization. Here, we examined the effect of CBLL1 knockdown and proteasome inhibitors on infection by WNV and other flaviviruses. We identified new siRNAs that repress the CBLL1 protein and strongly inhibit the endocytosis of Listeria monocytogenes, a bacterial pathogen known to require CBLL1 to invade host cells. Strikingly, however, we detected efficient WNV, dengue virus, and yellow fever virus infection of human cells, despite potent downregulation of CBLL1 by RNA interference. In addition, we found that the proteasome inhibitors MG132 and lactacystin did not affect WNV internalization but strongly repressed flavivirus RNA translation and replication. Together, these data do not support a requirement for CBLL1 during flavivirus entry and rather suggest an essential role of the ubiquitin/proteasome pathway for flavivirus genome amplification.
authors.affiliation: INSERM U944, Laboratoire de Pathologie et Virologie Moléculaire, Hôpital Saint-Louis, 1 Avenue Vellefaux, 75010 Paris, France.
authors.forename: Maria-Dolores
authors.initials: MD
authors.lastname: Fernandez-Garcia
authors.forename: Laurent
authors.initials: L
authors.lastname: Meertens
authors.forename: Matteo
authors.initials: M
authors.lastname: Bonazzi
authors.forename: Pascale
authors.initials: P
authors.lastname: Cossart
authors.forename: Fernando
authors.initials: F
authors.lastname: Arenzana-Seisdedos
authors.forename: Ali
authors.initials: A
authors.lastname: Amara
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000378:metabolism; D003716:Dengue Virus / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000378:metabolism; D008089:Listeria monocytogenes / Q000472:pathogenicity; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053586:Virus Internalization; D014779:Virus Replication; D014902:West Nile virus / Q000502:physiology*; D015005:Yellow fever virus / Q000502:physiology*
chemical_list: D007976:Leupeptins; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering; D025801:Ubiquitin; C067713:lactacystin; C471638:CBLL1 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine