2000
DOI: 10.1128/jvi.74.12.5556-5561.2000
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Characterization of Influenza Virus NS1 Protein by Using a Novel Helper-Virus-Free Reverse Genetic System

Abstract: We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NSdigested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This… Show more

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Cited by 26 publications
(27 citation statements)
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“…The sequence data provided interesting insights about the relevance for virus infectivity of the various protein regions of NS1. Thus, most of the mutations concentrated in the Nterminal half of the protein, which is known to contain a double-stranded RNA-binding domain (37,43) and to be sufficient to provide virus infectivity (12,14,17,63). However, mutations also appeared in the C-terminal region, proposed to constitute an effector domain (57).…”
Section: Discussionmentioning
confidence: 99%
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“…The sequence data provided interesting insights about the relevance for virus infectivity of the various protein regions of NS1. Thus, most of the mutations concentrated in the Nterminal half of the protein, which is known to contain a double-stranded RNA-binding domain (37,43) and to be sufficient to provide virus infectivity (12,14,17,63). However, mutations also appeared in the C-terminal region, proposed to constitute an effector domain (57).…”
Section: Discussionmentioning
confidence: 99%
“…Although it is not essential for virus viability (23), mutation in NS1 protein leads to defective viruses, some of which show temperature-sensitive phenotypes (12,14,17,27,28,40,60,(63)(64)(65). In addition, NS1 mutants are less virulent than wt viruses in experimental infections in animals (2,15,23,68).…”
Section: Discussionmentioning
confidence: 99%
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“…After 24 h, the cells were treated with IFN for 1 h and whole cell extracts were isolated in lysis buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 1 mM EDTA, 2.5 mM, 0.1% NP-40, 10% glycerol, 1 mM PMSF, and 10 lg/mL each of leupeptin, antipain, aprotinin, and pepstatin). Transfected NS1 was detected by immunofluorescence staining and western blotting using a polyclonal Ab against NS1 protein as described previously [67], and kindly provided by Dr. Masahiro Enami (Kanazawa University School of Medicine). Blots were also hybridized with a mouse anti-a-Tubulin mAb (1:2000; Sigma Chemical Co.).…”
Section: Ns1 Expression Plasmid and Transfectionmentioning
confidence: 99%