Browsing by Subject "Influenza A virus"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Battle between influenza A virus and a newly identified ZAPL antiviral activity(2015-05) Liu, Chien-Hung; Krug, Robert M.; Huibregtse, Jon M; Russell, Rick; Sullivan, Christopher S; Upton, JasonInfluenza A virus infection causes a highly contagious annual respiratory disease in humans as well as periodic pandemics with higher mortality rates. The Krug laboratory has shown that one of the major ways that the influenza virus NS1 protein counteracts host antiviral responses is to bind the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30). As a consequence, 3’ end processing of cellular pre-mRNAis is inhibited, leading to reduced production of cellular mRNAs, including interferon mRNAs. I showed that NS1-CPSF30 complexes contain an array of cellular proteins. I purified the NS1-CPSF30 complexes from virus infected cells by affinity selection of CPSF30 and the NS1 protein. I identified the associated cellular proteins by mass spectrometry. Two cellular RNA helicases, DDX21 and DHX30, were identified. SiRNA knockdown of either RNA helicase enhanced virus replication, indicating that DDX21 and DHX30 inhibit influenza A virus replication. Further study demonstrated that DDX21 RNA helicase inhibits viral RNA synthesis, and is countered by the NS1 protein. The cellular ZAPL antiviral protein was also identified in the NS1-CPSF30 complexes. Previous studies have shown that ZAPL antiviral activity is mediated by its N-terminal zinc-fingers, which targets viral mRNA of several viruses for degradation. Little is known about the antiviral role of the ZAPL C-terminal PARP domain. Here I discovered the antiviral role of ZAPL C-terminal PARP domain against influenza A virus. I showed that the ZAPL PARP domain targets the viral polymerase PA and PB2 proteins. These two viral polymerases are poly(ADP-ribosylated), presumably by other PARP protein(s). The ZAPL-associated, poly(ADP-ribosylated) PA and PB2 are then ubiquitinated and proteasomally degraded. This ZAPL antiviral activity is counteracted by the binding of polymerase PB1 protein to the WWE region adjacent to the PARP domain, and causes PA and PB2 to dissociate from ZAPL and thus escape degradation. Because PB1 displaces PA and PB2 and protects them from ZAPL-mediated degradation, endogenous ZAPL only moderately inhibits influenza A virus replication (20-30-fold), as determined by siRNA knockdown experiment. These results suggest that influenza A virus has partially won the battle against the newly identified ZAPL antiviral activity.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.