The role of the influenza NS1A protein during influenza A virus infection: evasion of the host anti-viral response
Year: 2005
Type: dissertation
Abstract: Influenza A viruses are a member of the family Orthomyxoviridae and are an \nimportant human pathogen causing wide spread disease and significant loss of life. The \nNS1A protein in influenza A virus plays a major role in blocking many cellular antiviral \nresponses. The effector domain of the NS1A protein, which comprises the C-terminal \ntwo-thirds of the protein, mediates the viral post-transcriptional countermeasure against \ncellular antiviral response through binding to the 30kDa subunit of CPSF, an essential \ncomponent of the cellular pre-mRNA 3’-end processing machinery. This binding inhibits \nthe post-transcriptional processing of cellular antiviral pre-mRNAs resulting in their \nnuclear accumulation and degradation. However, the function of its N-terminal RNAbinding \ndomain has not been established. To determine the function of the RNA-binding \ndomain of NS1A protein in virus infected cells, the recombinant influenza A virus \nencoding an NS1A protein lacking the binding site for dsRNA was generated. Analysis of \nthe phosphorylations of PKR and eIF-2α in this mutant virus infected cells established \nthat the dsRNA binding ability of NS1A is not required for blocking PKR activation in \nvivo. To determine whether the RNA-binding domain of NS1A protein is required for the \nresistance to the action of the interferon (IFN) in virus infected cells, an in vivo assay to \ndetermine the IFN sensitivity of viruses was developed. The IFN treatment caused \nattenuation of the dsRNA binding defective mutant virus, but not wild type virus, in \nsingle cycle growth indicating that dsRNA binding ability of the NS1A is required for the \nresistance to the action of the IFN in infected cells. The same assay after RNaseL downregulation \nusing RNA interference established that the resistance to IFN is mediated by \nblocking activation of [2’-5’ (A)] synthetase pathway. Since blocking PKR activation is \nnot mediated by the RNA-binding domain of NS1A protein we determined whether \nanother region of this protein is required for the inhibition of PKR activation. Serial \nmutagenesis experiments showed that amino acid residues 123 to 127 of NS1A protein \nare required for binding to PKR and the inhibition of its activation. Experiments using the \nPKR binding deficient mutant viruses revealed that the NS1A binding to PKR through \namino acid residues 123 to 127 is necessary and sufficient for blocking PKR activation \nduring influenza A virus infection. The activation of PKR in the mutant virus infected \ncells caused the inhibition of viral protein synthesis in virus infected cells. Moreover, the \nsynthesis of viral mRNA was greatly enhanced at earlier times of this mutant virus \ninfection, suggesting a functional interaction of the NS1A with the viral RNA polymerase \nthrough this region.
Author: Ji-Young Min
Language: English
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1887294564