YRKL sequence of influenza virus M1 functions as the L domain motif and interacts with VPS28 and Cdc42.
Earlier studies have shown that the C-terminal half of helix 6 (H6) of the influenza A virus matrix protein (M1) containing the YRKL sequence is involved in virus budding (E. K.-W. Hui, S. Barman, T. Y. Yang, and D. P. Nayak, J. Virol. 77:7078-7092, 2003). In this report, we show that the YRKL sequence is the L domain motif of influenza virus. Like other L domains, YRKL can be inserted at different locations on the mutant M1 protein and can restore virus budding in a position-independent manner. Although YRKL is a part of the nuclear localization signal (NLS), the function of YRKL was independent of the NLS activity and the NLS function of M1 was not required for influenza virus replication. Some mutations in YRKL and the adjacent region caused a reduction in the virus titer by blocking virus release, and some affected virus morphology, producing elongated particles. Coimmunoprecipitation and Western blotting analyses showed that VPS28, a component of the ESCRT-I complex, and Cdc42, a member of the Rho family GTP-binding proteins, interacted with the M1 protein via the YRKL motif. In addition, depletion of VPS28 and Cdc42 by small interfering RNA resulted in reduction of influenza virus production. Moreover, overexpression of dominant-negative Cdc42 inhibited influenza virus replication, whereas a constitutively active Cdc42 mutant enhanced virus production in infected cells. These results indicated that VPS28, a component of ESCRT-I, and Cdc42, a small G protein, are associated with the M1 protein and involved in the influenza virus life cycle.
Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, David Geffen School of Medicine, UCLA, Los Angeles, 90095, USA.
Eric Ka-Wai
EK
Hui
Subrata
S
Barman
Dominic Ho-Ping
DH
Tang
Bryan
B
France
Debi P
DP
Nayak
D020816:Amino Acid Motifs / Q000502:physiology*; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D056827:Endosomal Sorting Complexes Required for Transport; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D008958:Models, Molecular; D020125:Mutation, Missense; D019913:Nuclear Localization Signals / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D034741:RNA, Small Interfering; D033921:Vesicular Transport Proteins; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D020690:GTPase-Activating Proteins; C488936:M1 protein, Influenza A virus; D019913:Nuclear Localization Signals; D034741:RNA, Small Interfering; C476409:VPS28 protein, human; D033921:Vesicular Transport Proteins; D014763:Viral Matrix Proteins; D020764:cdc42 GTP-Binding Protein