Artificial Hybrids of Influenza A Virus RNA Polymerase Reveal PA Subunit Modulates Its Thermal Sensitivity

Kashiwagi, Takanari; Hara, Koyu; Nakazono, Yoko; Hamada, Nobuyuki; Watanabe, Hiroshi

doi:10.1371/journal.pone.0015140

Cited by 19 publications

(29 citation statements)

References 47 publications

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“…Through reassortant RNP molecules, they discovered that this activity is attributable to the PB2 gene (17). Kashiwagi et al also used VN04 and WSN reassortant RNP complexes, but they showed that thermal sensitivity is dependent on the species of origin of the PA gene (18). Although neither study investigated complexes containing the ts signature, our study is consistent with the findings of BradelTretheway et al and extends those findings by using infectious viruses.…”

Section: Discussionsupporting

confidence: 90%

The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

Broadbent

Santos

Godbout

et al. 2014

J Virol

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Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (>100-fold) replication at 39°C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. ts sig was restricted >100-fold compared to that at 33°C. Reassortant CA07 ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication >100-fold at 39°C. In ferrets, the CA07 ts sig virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07 ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. IMPORTANCELive attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses.

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Section: Discussionsupporting

confidence: 90%

The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

Broadbent

Santos

Godbout

et al. 2014

J Virol

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“…In addition to PB2, mutations in PA subunit have been reported to affect viral RNA replication [28]. Our results provided further evidence that the H1N1pdm09 PA dramatically enhanced viral polymerase activity of H5N1 at 33°C.…”

Section: Discussionsupporting

confidence: 69%

Replication and Transcription Activities of Ribonucleoprotein Complexes Reconstituted from Avian H5N1, H1N1pdm09 and H3N2 Influenza A Viruses

Ngai

Chan

2013

PLoS ONE

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Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.

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“…It has also been suggested that zoonotic transmission may be limited by temperature differences between the two hosts [3]. In this regard, we compared the polymerase activities of the recombinant vRNPs at 33°C and 37°C.…”

Section: Discussionmentioning

confidence: 99%

Polymerase activity of hybrid ribonucleoprotein complexes generated from reassortment between 2009 pandemic H1N1 and seasonal H3N2 influenza A viruses

Lam

Ngai

Chan

2011

Virol J

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BackgroundA novel influenza virus (2009 pdmH1N1) was identified in early 2009 and progressed to a pandemic in mid-2009. This study compared the polymerase activity of recombinant viral ribonucleoprotein (vRNP) complexes derived from 2009 pdmH1N1 and the co-circulating seasonal H3N2, and their possible reassortants.ResultsThe 2009 pdmH1N1 vRNP showed a lower level of polymerase activity at 33°C compared to 37°C, a property remenisence of avian viruses. The 2009 pdmH1N1 vRNP was found to be more cold-sensitive than the WSN or H3N2 vRNP. Substituion of 2009 pdmH1N1 vRNP with H3N2-derived-subunits, and vice versa, still retained a substantial level of polymerase activity, which is probably compartable with survival. When the 2009 pdmH1N1 vRNP was substituted with H3N2 PA, a significant increase in activity was observed; whereas when H3N2 vRNP was substituted with 2009 pdmH1N1 PA, a significant decrease in activity occurred. Although, the polymerase basic protein 2 (PB2) of 2009 pdmH1N1 was originated from an avian virus, substitution of this subunit with H3N2 PB2 did not change its polymerase activity in human cells.ConclusionsIn conclusion, our data suggest that hybrid vRNPs resulted from reassortment between 2009 pdmH1N1 and H3N2 viruses could still retain a substantial level of polymerase activity. Substituion of the subunit PA confers the most prominent effect on polymerase activity. Further studies to explore the determinants for polymerase activity of influenza viruses in associate with other factors that limit host specificity are warrant.

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Artificial Hybrids of Influenza A Virus RNA Polymerase Reveal PA Subunit Modulates Its Thermal Sensitivity

Cited by 19 publications

References 47 publications

The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

Replication and Transcription Activities of Ribonucleoprotein Complexes Reconstituted from Avian H5N1, H1N1pdm09 and H3N2 Influenza A Viruses

Polymerase activity of hybrid ribonucleoprotein complexes generated from reassortment between 2009 pandemic H1N1 and seasonal H3N2 influenza A viruses

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