DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Functional Analysis of Conserved Motifs in Influenza Virus PB1 Protein

Abstract

The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assaysmore » suggests compensatory mutations and/or mixed infections which may have ‘rescued’ the inactive PB1 protein.« less

Authors:
 [1];  [1];  [1];  [2];  [1];  [1];  [1];  [3]
  1. University of Wisconsin, Madison, WI (United States)
  2. Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
  3. Univ. of Wisconsin, Madison, WI (United States); University of Tokyo (Japan)
Publication Date:
Research Org.:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); Pfizer Animal Health
OSTI Identifier:
1627511
Grant/Contract Number:  
AC52-06NA25396
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 7; Journal Issue: 5; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; polymerases; microbial mutation; sequence motif analysis; H5N1; influenza A virus; viral replication; influenza viruses; H1N1

Citation Formats

Chu, Caroline, Fan, Shufang, Li, Chengjun, Macken, Catherine, Kim, Jin Hyun, Hatta, Masato, Neumann, Gabriele, and Kawaoka, Yoshihiro. Functional Analysis of Conserved Motifs in Influenza Virus PB1 Protein. United States: N. p., 2012. Web. doi:10.1371/journal.pone.0036113.
Chu, Caroline, Fan, Shufang, Li, Chengjun, Macken, Catherine, Kim, Jin Hyun, Hatta, Masato, Neumann, Gabriele, & Kawaoka, Yoshihiro. Functional Analysis of Conserved Motifs in Influenza Virus PB1 Protein. United States. https://doi.org/10.1371/journal.pone.0036113
Chu, Caroline, Fan, Shufang, Li, Chengjun, Macken, Catherine, Kim, Jin Hyun, Hatta, Masato, Neumann, Gabriele, and Kawaoka, Yoshihiro. Tue . "Functional Analysis of Conserved Motifs in Influenza Virus PB1 Protein". United States. https://doi.org/10.1371/journal.pone.0036113. https://www.osti.gov/servlets/purl/1627511.
@article{osti_1627511,
title = {Functional Analysis of Conserved Motifs in Influenza Virus PB1 Protein},
author = {Chu, Caroline and Fan, Shufang and Li, Chengjun and Macken, Catherine and Kim, Jin Hyun and Hatta, Masato and Neumann, Gabriele and Kawaoka, Yoshihiro},
abstractNote = {The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assays suggests compensatory mutations and/or mixed infections which may have ‘rescued’ the inactive PB1 protein.},
doi = {10.1371/journal.pone.0036113},
journal = {PLoS ONE},
number = 5,
volume = 7,
place = {United States},
year = {Tue May 15 00:00:00 EDT 2012},
month = {Tue May 15 00:00:00 EDT 2012}
}

Works referenced in this record:

Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses
journal, May 2000


Towards an atomic resolution understanding of the influenza virus replication machinery
journal, February 2010

  • Ruigrok, Rob WH; Crépin, Thibaut; Hart, Darren J.
  • Current Opinion in Structural Biology, Vol. 20, Issue 1
  • DOI: 10.1016/j.sbi.2009.12.007

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.
journal, December 1989


Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses
journal, January 1984


Evolution and ecology of influenza A viruses.
journal, January 1992


In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses
journal, July 2009

  • Itoh, Yasushi; Shinya, Kyoko; Kiso, Maki
  • Nature, Vol. 460, Issue 7258
  • DOI: 10.1038/nature08260

Efficient selection for high-expression transfectants with a novel eukaryotic vector
journal, December 1991


Characterization of influenza virus RNA complete transcripts
journal, January 1982


Generation of influenza A viruses entirely from cloned cDNAs
journal, August 1999

  • Neumann, G.; Watanabe, T.; Ito, H.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 16
  • DOI: 10.1073/pnas.96.16.9345

A sequence motif in many polymerases
journal, January 1988


Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA
journal, April 2011


Genetic Engineering of Influenza and Other Negative-Strand RNA Viruses Containing Segmented Genomes
book, January 1999


Characterization of influenza virus RNA complete transcripts
journal, January 1982


Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA
journal, April 2011


Towards an atomic resolution understanding of the influenza virus replication machinery
journal, February 2010

  • Ruigrok, Rob WH; Crépin, Thibaut; Hart, Darren J.
  • Current Opinion in Structural Biology, Vol. 20, Issue 1
  • DOI: 10.1016/j.sbi.2009.12.007

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses
journal, July 2009

  • Itoh, Yasushi; Shinya, Kyoko; Kiso, Maki
  • Nature, Vol. 460, Issue 7258
  • DOI: 10.1038/nature08260

Reverse genetics of negative-strand RNA viruses: Closing the circle
journal, August 1999

  • Pekosz, Andrew; He, Biao; Lamb, Robert A.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 16
  • DOI: 10.1073/pnas.96.16.8804

Influenza A Virus Polymerase: Structural Insights into Replication and Host Adaptation Mechanisms
journal, September 2010

  • Boivin, Stéphane; Cusack, Stephen; Ruigrok, Rob W. H.
  • Journal of Biological Chemistry, Vol. 285, Issue 37
  • DOI: 10.1074/jbc.r110.117531

Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses
journal, January 1984


A sequence motif in many polymerases
journal, January 1988


Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses
journal, May 2000


Properties and Dissemination of H5N1 Viruses Isolated during an Influenza Outbreak in Migratory Waterfowl in Western China
journal, June 2006

  • Chen, Hualan; Li, Yanbing; Li, Zejun
  • Journal of Virology, Vol. 80, Issue 12
  • DOI: 10.1128/jvi.00110-06

Evidence for Avian and Human Host Cell Factors That Affect the Activity of Influenza Virus Polymerase
journal, July 2010

  • Moncorgé, Olivier; Mura, Manuela; Barclay, Wendy S.
  • Journal of Virology, Vol. 84, Issue 19
  • DOI: 10.1128/jvi.01134-10

Determination of influenza virus proteins required for genome replication.
journal, January 1990


Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1.
journal, January 1994


Differential activation of the influenza virus polymerase via template RNA binding.
journal, January 1995


Direct Evidence that the Poly(A) Tail of Influenza A Virus mRNA Is Synthesized by Reiterative Copying of a U Track in the Virion RNA Template
journal, April 1999


Influenza A Virus RNA Polymerase Has the Ability To Stutter at the Polyadenylation Site of a Viral RNA Template during RNA Replication
journal, June 1999


Cloning of the Chicken RNA Polymerase I Promoter and Use for Reverse Genetics of Influenza A Viruses in Avian Cells
journal, October 2005


Evolution and ecology of influenza A viruses
journal, March 1992


Works referencing / citing this record:

The RNA-dependent RNA polymerase of the influenza A virus
journal, September 2014

  • Stubbs, Thomas M.; te Velthuis, Aartjan JW
  • Future Virology, Vol. 9, Issue 9
  • DOI: 10.2217/fvl.14.66

Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution
journal, August 2017


Common and unique features of viral RNA-dependent polymerases
journal, August 2014


Common and unique features of viral RNA-dependent polymerases.
text, January 2014


Amino Acid Changes in the Influenza A Virus PA Protein That Attenuate Avian H5N1 Viruses in Mammals
journal, September 2014


Amino Acid Substitutions in PB1 of Avian Influenza Viruses Influence Pathogenicity and Transmissibility in Chickens
journal, July 2014

  • Suzuki, Y.; Uchida, Y.; Tanikawa, T.
  • Journal of Virology, Vol. 88, Issue 19
  • DOI: 10.1128/jvi.01564-14

High-throughput profiling of influenza A virus hemagglutinin gene at single-nucleotide resolution
journal, May 2014

  • Wu, Nicholas C.; Young, Arthur P.; Al-Mawsawi, Laith Q.
  • Scientific Reports, Vol. 4, Issue 1
  • DOI: 10.1038/srep04942

Integrating computational modeling and functional assays to decipher the structure-function relationship of influenza virus PB1 protein
journal, November 2014

  • Li, Chunfeng; Wu, Aiping; Peng, Yousong
  • Scientific Reports, Vol. 4, Issue 1
  • DOI: 10.1038/srep07192

Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice
journal, February 2016

  • Feng, Xiaoxiao; Wang, Zeng; Shi, Jianzhong
  • Journal of Virology, Vol. 90, Issue 4
  • DOI: 10.1128/jvi.02387-15

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis
journal, December 2016


Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution
journal, August 2017


Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality
journal, July 2015


Common and unique features of viral RNA-dependent polymerases.
text, January 2014