Mutational Analysis of Conserved Amino Acids in the Influenza A Virus Nucleoprotein

Li, Zejun; Watanabe, Tokiko; Hatta, Masato; Watanabe, Shinji; Nanbo, Asuka; Ozawa, Makoto; Kakugawa, Satoshi; Shimojima, Masayuki; Yamada, S.; Neumann, Gabriele; Kawaoka, Yoshihiro

doi:10.1128/jvi.02642-08

Cited by 99 publications

(110 citation statements)

References 41 publications

(28 reference statements)

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“…All the NP substitutions we identified (except M159I, which is linked to A336T) cluster in a region thought to influence NP-PB2 interactions (27,32), suggesting a common function. While all NP sub- stitutions are at highly conserved positions in natural isolates, A336T and F346S in particular are adjacent to variable positions.…”

Section: Discussionmentioning

confidence: 99%

Reassortment Complements Spontaneous Mutation in Influenza A Virus NP and M1 Genes To Accelerate Adaptation to a New Host

Ince

Gueye-Mbaye

Bennink

et al. 2013

J Virol

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Influenza A virus (IAV) infects a remarkably wide variety of avian and mammalian hosts. Evolution finely hones IAV genes to optimally infect and be transmitted in a particular host species. Sporadically, IAV manages to jump between species, introducing novel antigenic strains into the new host population that wreak havoc until herd immunity develops. IAV adaptation to new hosts typically involves reassortment of IAV gene segments from coinfecting virus strains adapted to different hosts in conjunction with multiple adaptive mutations in the various IAV genes. To better understand host adaptation between mammalian species in real time, we passaged mouse-adapted A/PR8/34 (PR8) in guinea pigs. Guinea pigs, unlike mice, support spontaneous and robust IAV transmission. For some IAV strains, including PR8, adaptation is required for a virus to attain transmissibility, providing an opportunity to understand the evolution of transmissibility in guinea pigs. Multiple guinea pig-adapted PR8 mutants generated by serial nasal wash passaging in independent lines replicated more efficiently and were transmitted by cocaging. All transmissible variants possessed one of two nonsynonymous mutations in M1, either alone or in combination with mutations in PB2, HA, NP, or NA. Rapid reassortment between independently selected variants combined beneficial mutations in NP and M1 to form the fittest virus capable of being transmitted. These findings provide further insight into genetic determinants in NP and M1 involved in PR8 IAV adaptation to be transmitted in a new host and clearly show the benefit of a segmented genome in rapidly generating optimal combinations of mutations in IAV evolution.

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Section: Discussionmentioning

confidence: 99%

Reassortment Complements Spontaneous Mutation in Influenza A Virus NP and M1 Genes To Accelerate Adaptation to a New Host

Ince

Gueye-Mbaye

Bennink

et al. 2013

J Virol

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“…cDNA was synthesized by using the SuperScript III first-strand synthesis system (Invitrogen). The primers for reverse transcription (RT) of mRNA, vRNA, and U2 small nuclear RNA (snRNA) were oligo(dT) 20 , an IAV-specific RT primer (uni-12; 5=-AGCAAAAGCAGG-3=), and a U2 snRNA-specific RT primer (5=-CTGGAGGTACTGCAATACCAG-3=), respectively. For most quantitative RT (qRT)-PCR analyses, we followed the standard TaqMan method with the Universal Probe Library System.…”

Section: Methodsmentioning

confidence: 99%

A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

Hsu

Lee

et al. 2015

J Virol

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Influenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals. IMPORTANCEInfluenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we reported that RRP1B is crucial for RdRp binding to cellular capped mRNA, which is a critical step of virus transcription. Our study not only provides a deeper understanding of influenza virus-host interplay, but also suggests a potential target for antiviral drug development.

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“…Many mutants of NP, including some tail-loop mutants, lose the ability to support the RDRP activity in reconstitution experiments (2,(15)(16)(17)(18). In addition, some of the mutants are shown to exist in monomers instead of trimers.…”

mentioning

confidence: 99%

E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors

Shen

Chen

Chu

et al. 2011

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

110

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The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism-disruption of NP-NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402-428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP-NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozinresistant strains. This work provides a new approach to design antiinfluenza drugs.T he RNA-dependent RNA polymerase (RDRP) of the influenza A virus is composed of polymerase basic protein 1 (PB1), basic protein 2 (PB2), and acidic protein (PA) (1). The function of RDRP for viral replication requires association with the nucleoprotein (NP) (2) to form the ribonucleoprotein (RNP) complex. Only low resolution structures of the RNP complex are available from cryo-EM studies (2-9), whereas high resolution structures have been reported for some individual components or fragments (10-12). Crystal structures of NP indicate that it exists in trimers (13,14), with the tail-loop (residues 402-428) region playing an important role in the trimerization (Fig. 1A). Based on the structural information, it was suggested that the tail-loop binding pocket could be a target for antiinfluenza therapeutics (13,14).Disruption of the NP-NP interaction as a strategy for designing antiinfluenza drugs has been further reported. Many mutants of NP, including some tail-loop mutants, lose the ability to support the RDRP activity in reconstitution experiments (2,(15)(16)(17)(18). In addition, some of the mutants are shown to exist in monomers instead of trimers. These results support the importance of NP in the RDRP activity and viral replication, and the possibility of NP as a drug target. However, it remains to be shown that molecules capable of disrupting the NP-NP interaction would inhibit viral replication.Recently Kao et al. (19) and our group (20) reported the use of high throughput screening to identify nucleozin and its analogs as inhibitors that halt viral replication by binding to NP and causing it...

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Mutational Analysis of Conserved Amino Acids in the Influenza A Virus Nucleoprotein

Cited by 99 publications

References 41 publications

Reassortment Complements Spontaneous Mutation in Influenza A Virus NP and M1 Genes To Accelerate Adaptation to a New Host

Reassortment Complements Spontaneous Mutation in Influenza A Virus NP and M1 Genes To Accelerate Adaptation to a New Host

A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors

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