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