نبذة مختصرة : Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.
Author summary Due to limited genome capacity, viral proteins often fulfill multiple functions during viral replication. Matrix proteins of enveloped viruses have to be intrinsically multifunctional, since they confer integrity of the virion, but also function in disintegration during virus entry. The highly conserved influenza A virus matrix protein 1 (M1) is a prototype for such a multifunctional protein, however, the underlying mechanisms that regulate its functional switches are only scarcely understood. Here, we unraveled M1 phosphorylation at tyrosine 132 as a spatio-temporal regulatory mechanism that potentially induces structural diversification and is needed for efficient viral genome incorporation during assembly. The site is most likely phosphorylated by cellular membrane-associated Janus kinases late in infection, suggesting that these kinases might serve as novel cellular targets for antiviral intervention.
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