Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication

Lutz, Andrew; Dyall, Julie; Olivo, Paul D.; Pekosz, Andrew

doi:10.1016/j.jviromet.2005.01.016

Cited by 96 publications

(105 citation statements)

References 44 publications

(47 reference statements)

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“…10 ng pRL-SV40 constitutively expressing Renilla luciferase was added to normalize transfection efficiency. It was previously shown that expression of the firefly luciferase reporter gene correlates with the activity of the reconstituted polymerase complex (45).…”

Section: Methodsmentioning

confidence: 99%

Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

Graf

Dick

Sendker

et al. 2018

Journal of Biological Chemistry

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Only a minority of patients infected with seasonal influenza A viruses exhibits a severe or fatal outcome of infection, but the reasons for this inter-individual variability in influenza susceptibility are unclear. To gain further insights into the molecular mechanisms underlying this variability, we investigated naturally occurring allelic variations of the myxovirus resistance 1 (MX1) gene coding for the influenza restriction factor MxA. The interferon-induced dynamin-like GTPase consists of an N-terminal GTPase domain, a bundle signaling element, and a C-terminal stalk responsible for oligomerization and viral target recognition. We used online databases to search for variations in the MX1 gene. Deploying in vitro approaches, we found that non-synonymous variations in the GTPase domain cause the loss of antiviral and enzymatic activities. Furthermore, we showed that these amino acid substitutions disrupt the interface for GTPase domain dimerization required for the stimulation of GTP hydrolysis. Variations in the stalk were neutral or slightly enhanced or abolished MxA antiviral function.http://www.jbc.org/cgi

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Section: Methodsmentioning

confidence: 99%

Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

Graf

Dick

Sendker

et al. 2018

Journal of Biological Chemistry

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“…The authors are grateful to Professor Dr Gert Zimmer (University of Hannover, Germany) for providing PKC expression constructs, Professor Dr Erich Hoffmann (UTMB, TX, USA) for providing a set of influenza virus expression constructs, the Company Apath LLC (St Louis, MO, USA) for providing the influenza GFP reporter construct pHH21-NP-UTRhi-eGFP (Lutz et al, 2005), Dr Klaus Korn and Helga Bischoff (Institute of Clinical and Molecular Virology, University of Erlangen-Nuremberg) for support with the influenzaspecific qRT-PCR and excellent technical assistance, respectively, Dr György Keri (Vichem Chemie Research Ltd, Budapest, Hungary) for supplying a panel of protein kinase inhibitors, Professor Dr Michael Stürzl and Andreas Konrad (Institute of Experimental Surgery, University of Erlangen-Nuremberg) for methodical collaboration, Sabine Rechter and Stephanie Platzer for help in establishing the influenza reporter system and IVKAs and Jens Milbradt for scientific discussion. This work was supported by the ELAN Fonds of the University of Erlangen-Nuremberg and the Deutsche Forschungsgemeinschaft (grant MA 1289/4-1).…”

Section: Acknowledgementsmentioning

confidence: 99%

“…Transient transfection of 293T cells was performed in 12-well plates at a confluence of 70-90 % (seeding cell number 2.5610 5 ) by using Lipofectamine 2000 (Invitrogen). The influenza-specific green fluorescent protein (GFP) construct pHH21-NP-UTRhi-eGFP (Lutz et al, 2005) served as a reporter of viral replication. One day post-transfection with the reporter construct, cells were infected with influenza A/WSN/33 virus and were subsequently cultivated in the presence or absence of Gö6976 (Fig.…”

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confidence: 99%

“…We used a set of constructs previously generated by Lutz et al (2005) and Hoffmann et al (2000) to perform with expression constructs for the FLAG (F)-tagged proteins, as indicated. Two days posttransfection, cells were lysed and proteins were immunoprecipitated by the use of mAb-FLAG (a and c) or mAb-FLAG plus mAb-GFP (b) for subsequent analysis in a PKC-specific IVKA.…”

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confidence: 99%

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Influenza A virus proteins PB1 and NS1 are subject to functionally important phosphorylation by protein kinase C

Mahmoudian

Auerochs

Gröne

et al. 2009

Journal of General Virology

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The virulence of influenza A viruses depends on the activity of the viral RNA polymerase complex and viral regulatory phosphoproteins. We identified that the protein kinase C (PKC) inhibitor Gö 6976 had a post-entry anti-influenza viral effect, by using a polymerase activity-based reporter assay. This inhibitory effect was observed for influenza virus-infected cells as well as for cells transiently transfected with constructs for the RNA polymerase complex. Importantly, the in vitro analysis of viral protein phosphorylation identified PKCa as a kinase phosphorylating PB1 and NS1, but not PB2, PA or NP. Gö 6976 was able to block PKC-specific phosphorylation in vitro. Thus, our data suggest that PKC contributes to the phosphorylation of influenza PB1 and NS1 proteins which appears to be functionally relevant for both viral RNA polymerase activity and efficient viral replication.Influenza A viruses are pathogens that cause significant morbidity and mortality in humans and a variety of animal species (Palese & Shaw, 2007;Wright et al., 2007). Influenza viruses possess a segmented negative-sense RNA genome which is replicated in the nucleus of infected cells. The virulence of individual virus strains is based on a complex co-action of determinants, including the viral haemagglutinin (HA), the RNA-dependent RNA polymerase (subunits PB1, PB2 and PA) and the nonstructural protein (NS1) (Geiss et al., 2002;Tumpey et al., 2005Tumpey et al., , 2007. The RNA-dependent RNA polymerase complex is responsible for both transcription and genomic replication (Torreira et al., 2007;Palese & Shaw, 2007). PB1 represents the central catalytic subunit of the polymerase complex (Honda & Ishihama, 1997;Poch et al., 1989;Torreira et al., 2007), PB2 is responsible for recognition and binding of the cap structures of host mRNAs which are used for priming viral transcription (Fechter & Brownlee, 2005;Guilligay et al., 2008), while PA combines essential activities required for genome replication and protein processing (Fodor & Smith, 2004;Maier et al., 2008;Palese & Shaw, 2007). The viral nucleoprotein (NP) strictly associates with the RNA polymerase complex to form viral ribonucleoproteins (RNPs) (Noda et al., 2006;Portela & Digard, 2002), it stimulates RNA polymerase activity and mediates nuclear RNP translocation (Neumann et al., 1997;Bui et al., 2002;Portela & Digard, 2002). The viral regulatory protein NS1 provides a number of functions required for efficient replication and gene expression (Hale et al., 2008a). An interaction between NS1 and the viral transcription-replication complex was reported (Marion et al., 1997) but awaits a confirmation in functional aspects. NS1 and the RNP components including NP are phosphoproteins in various types and strains of influenza viruses (Sanz-Ezquerro et al., 1998;Bui et al., 2002;Arrese & Portela, 1996;Skorko et al., 1991;Marschall et al., 1999;Neumann et al., 1997;Privalsky & Penhoet, 1978, 1981. Several cellular protein kinases, including protein kinase C (PKC), casein kinase II, cyclin-dependen...

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“…1B). We explored the inhibition activities of the antiinfluenza compounds using three additional assays: influenza neuraminidase, influenza entry using the pseudotyped virus expressing influenza hemagglutinin, HApp (17), and the cell-based reporter assay for the influenza RdRP (18). Except for the known neuraminidase inhibitors Oseltamivir and Zanamivir, no other compounds are inhibitory to the influenza neuraminidase activity.…”

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confidence: 99%

High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

Cheng

Lin

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.high-throughput screening | antiinfluenza | influenza NP | influenza PB1 | chemical genetics

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Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication

Cited by 96 publications

References 44 publications

Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

Influenza A virus proteins PB1 and NS1 are subject to functionally important phosphorylation by protein kinase C

High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

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