Browsing by Subject "Influenza viruses"
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Item Influenza virus polymerases: determination of the cap binding site and the crucial role of CA endonuclease cleavage site in the cap snatching mechanism for the initiation of viral messenger RNA synthesis(2003) Rao, Ping; Krug, Robert M.It has been established that influenza virus synthesizes its viral mRNAs through a "cap-snatching" mechanism; specifically, the viral mRNA synthesis is initiated with capped-RNA primers generated by the endonuclease intrinsic to the viral polymerase from host cellular mRNAs. To exert the cap-binding and endonuclease activity, the polymerase must be activated by the conserved terminal sequences in the viral genomic RNAs (vRNAs). By UV cross-linking and protein microsequencing, a short peptide (544-556 in the PB2 protein) is found to directly bind to the capped-RNAs. To identify the specific amino acids involved in direct cap recognition, five aromatic amino acids around this region were mutated individually to alanine. Four of the five mutated PB2 proteins are unable to form functional polymerase complexes with other subunits. The fifth mutant, W552A, which forms functional polymerase, has attenuated cap-binding and endonuclease activities. Therefore, some of the other four aromatic amino acids may be involved in direct cap recognition. Using different capped RNA as substrates for endonuclease, I compared the effect of the dinucleotides at the cleavage site on the endonuclease activity and transcription initiation in vitro. I demonstrate that only the substrate containing a 3' CA terminus is effectively used as a primer for viral mRNA synthesis. This explains the previous observation that the vast majority of cellular capped primers used during virus infection contain CA at their 3' termini. Using this system, I found, in contrast to previous reports, the 5' terminal sequence of viral RNA (vRNA) alone is sufficient for the activation of the endonuclease, and the 3' terminal sequence of vRNA functions solely as template for transcription initiation. I further identified the sequence/structure in the 5' and 3' termini of vRNA that are both necessary and sufficient for their function as endonuclease activator and transcription template, respectively. The 3'vRNA binds the polymerase by both RNA-polymerase and RNA-5’vRNA interactions. My results also indicate that the viral mRNA elongation may be bi-phasic. In early elongation, within 18 nucleotides from the transcription initiation site, the viral polymerase and the conserved vRNA terminal sequences are sufficient. In the second phase of elongation, additional factors/conditions are required.Item The role of the influenza NS1A protein during influenza A virus infection: evasion of the host anti-viral response(2005) Min, Ji-Young; Krug, Robert M.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.Item The role of the non-structural protein of human influenza A viruses (NS1A protein) during infection of human cells(2002-08) Kim, Mee-jung; Krug, Robert M.