Browsing by Subject "Virus inhibitors"
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Item The NS1A protein of influenza A virus: its crucial role in the inhibition of 3' end processing of cellular pre-mRNAs(2006) Twu, Karen Yuan-Yun; Krug, Robert M.Influenza A viruses, one of the four influenza virus genera, are responsible for the human pandemics that have caused high mortality rates, and the highly pathogenic virus transmitted directly from chickens to humans in 1997 is an influenza A virus. It has been shown previously that influenza A viruses, like many mammalian viruses induce the interferon-α/β (IFN-α/β)-independent activation of interferon regulatory factor-3 (IRF-3) and transcription of some IFN-α/β–stimulated response element (ISRE)-controlled cellular genes. This cellular defense against virus infection takes place prior to the synthesis of IFN-α/β. In addition, influenza A viruses are unique in that their nonstructural protein (NS1A protein) inhibits the posttranscriptional processing of these cellular antiviral pre-mRNAs. The NS1A protein contains two functional domains: an RNA-binding/dimerization domain at the amino terminus and an effector domain in the carboxyl half. The effector domain physically associates with two essential components of the machinery of the 3′ end processing of cellular pre-mRNAs: the 30kDa subunit of the cleavage and polyadenylation specificity factor (CPSF) and poly(A)-binding protein (PABII). The consequent inhibition of CPSF and PABII function inhibits the 3′ end processing of cellular pre-mRNAs in virus-infected cells, and as a result, cellular premRNAs are not cleaved and nuclear export of mature mRNAs is inhibited. This action of the NS1A protein also inhibits the production of IFN-β mRNA. Binding of CPSF30 to NS1A is mediated by two of its zinc finger domains, F2 and F3, and the CPSF30/F2F3 binding site on the NS1A protein extending from amino acid 144 to 186. We generated MDCK cells that constitutively express epitope-tagged F2F3, and showed that influenza A virus replication was selectively inhibited in this cell line. Influenza A virus induced increased production of IFN-β mRNA in the F2F3-expressing cells. These results, which indicate that F2F3 inhibits influenza A virus replication by blocking the binding of endogenous CPSF30 to the NS1A protein, point to this NS1A binding site as a potential target for the development of antivirals directed against influenza A virus. Moreover, we found the NS1A protein encoded by HK/483/97, the initial H5N1 virus that was transmitted from chicken to human in 1997, does not inhibit posttranscriptional processing of cellular pre-mRNAs. As a consequence, a chimeric Udorn/H5N1 virus that contains only the NS gene from A/HK/483/97 virus induced the production of a high level of IFN-β mRNA and was highly attenuated. In contrast, the NS1A protein encoded by the pathogenic H5N1 virus isolated in 2004 inhibited pre-mRNA processing, resulting in decreased production of IFN-β mRNA. These results demonstrate that the NS1A protein in H5N1 viruses acquired a function between 1997 and 2004 that enhances virus replication in mammalian cells.Item Production and characterization of bovine interferons(Texas Tech University, 1979-08) Luk, Alvin Dip-HingNot availableItem Production and characterization of murine interferons(Texas Tech University, 1979-08) Tyring, Stephen KeithNot availableItem 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.