The role of the influenza NS1A protein during influenza A virus infection: evasion of the host anti-viral response

Influenza A viruses are a member of the family Orthomyxoviridae and are an important human pathogen causing wide spread disease and significant loss of life. The NS1A protein in influenza A virus plays a major role in blocking many cellular antiviral responses. The effector domain of the NS1A protein, which comprises the C-terminal two-thirds of the protein, mediates the viral post-transcriptional countermeasure against cellular antiviral response through binding to the 30kDa subunit of CPSF, an essential component of the cellular pre-mRNA 3’-end processing machinery. This binding inhibits the post-transcriptional processing of cellular antiviral pre-mRNAs resulting in their nuclear accumulation and degradation. However, the function of its N-terminal RNAbinding domain has not been established. To determine the function of the RNA-binding domain of NS1A protein in virus infected cells, the recombinant influenza A virus encoding an NS1A protein lacking the binding site for dsRNA was generated. Analysis of the phosphorylations of PKR and eIF-2α in this mutant virus infected cells established that the dsRNA binding ability of NS1A is not required for blocking PKR activation in vivo. To determine whether the RNA-binding domain of NS1A protein is required for the resistance to the action of the interferon (IFN) in virus infected cells, an in vivo assay to determine the IFN sensitivity of viruses was developed. The IFN treatment caused attenuation of the dsRNA binding defective mutant virus, but not wild type virus, in single cycle growth indicating that dsRNA binding ability of the NS1A is required for the resistance to the action of the IFN in infected cells. The same assay after RNaseL downregulation using RNA interference established that the resistance to IFN is mediated by blocking activation of [2’-5’ (A)] synthetase pathway. Since blocking PKR activation is not mediated by the RNA-binding domain of NS1A protein we determined whether another region of this protein is required for the inhibition of PKR activation. Serial mutagenesis experiments showed that amino acid residues 123 to 127 of NS1A protein are required for binding to PKR and the inhibition of its activation. Experiments using the PKR binding deficient mutant viruses revealed that the NS1A binding to PKR through amino acid residues 123 to 127 is necessary and sufficient for blocking PKR activation during influenza A virus infection. The activation of PKR in the mutant virus infected cells caused the inhibition of viral protein synthesis in virus infected cells. Moreover, the synthesis of viral mRNA was greatly enhanced at earlier times of this mutant virus infection, suggesting a functional interaction of the NS1A with the viral RNA polymerase through this region.