RESUMO
Lysine acetylation is a post-translational modification known to regulate protein functions. Here we identify several acetylation sites of the influenza A virus nucleoprotein (NP), including the lysine residues K77, K113 and K229. Viral growth of mutant virus encoding K229R, mimicking a non-acetylated NP lysine residue, is severely impaired compared to wildtype or the mutant viruses encoding K77R or K113R. This attenuation is not the result of decreased polymerase activity, altered protein expression or disordered vRNP co-segregation but rather caused by impaired particle release. Interestingly, release deficiency is also observed mimicking constant acetylation at this site (K229Q), whereas virus encoding NP-K113Q could not be generated. However, mimicking NP hyper-acetylation at K77 and K229 severely diminishes viral polymerase activity, while mimicking NP hypo-acetylation at these sites has no effect on viral replication. These results suggest that NP acetylation at K77, K113 and K229 impacts multiple steps in viral replication of influenza A viruses.
Assuntos
Vírus da Influenza A/genética , Lisina/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/genética , Replicação Viral/genética , Acetilação , Animais , Cães , Células HEK293 , Humanos , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/metabolismo , Células Madin Darby de Rim Canino , Mutação , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismoRESUMO
To establish a new lineage in the human population, avian influenza A viruses (AIV) must overcome the intracellular restriction factor MxA. Partial escape from MxA restriction can be achieved when the viral nucleoprotein (NP) acquires the critical human-adaptive amino acid residues 100I/V, 283P, and 313Y. Here, we show that introduction of these three residues into the NP of an avian H5N1 virus renders it genetically unstable, resulting in viruses harboring additional single mutations, including G16D. These substitutions restored genetic stability yet again yielded viruses with varying degrees of attenuation in mammalian and avian cells. Additionally, most of the mutant viruses lost the capacity to escape MxA restriction, with the exception of the G16D virus. We show that MxA escape is linked to attenuation by demonstrating that the three substitutions promoting MxA escape disturbed intracellular trafficking of incoming viral ribonucleoprotein complexes (vRNPs), thereby resulting in impaired nuclear import, and that the additional acquired mutations only partially compensate for this import block. We conclude that for adaptation to the human host, AIV must not only overcome MxA restriction but also an associated block in nuclear vRNP import. This inherent difficulty may partially explain the frequent failure of AIV to become pandemic.
Assuntos
Substituição de Aminoácidos , Virus da Influenza A Subtipo H5N1/genética , Proteínas de Resistência a Myxovirus/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Células A549 , Animais , Aves/virologia , Linhagem Celular , Cães , Células HEK293 , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação , Proteínas do Nucleocapsídeo , Conformação Proteica , Transporte Proteico , Proteínas de Ligação a RNA/química , Proteínas do Core Viral/químicaRESUMO
Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A(1-25)) with that of FluB (PB1-B(1-25)) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B(1-25) peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A(1-25) peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.