PMID- 20454455
OWN - NLM
STAT- MEDLINE
DCOM- 20110616
LR  - 20211020
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 5
IP  - 4
DP  - 2010 Apr 29
TI  - Biochemical characterization of enzyme fidelity of influenza A virus RNA 
      polymerase complex.
PG  - e10372
LID - 10.1371/journal.pone.0010372 [doi]
LID - e10372
AB  - BACKGROUND: It is widely accepted that the highly error prone replication process 
      of influenza A virus (IAV), together with viral genome assortment, facilitates 
      the efficient evolutionary capacity of IAV. Therefore, it has been logically 
      assumed that the enzyme responsible for viral RNA replication process, influenza 
      virus type A RNA polymerase (IAV Pol), is a highly error-prone polymerase which 
      provides the genomic mutations necessary for viral evolution and host adaptation. 
      Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never 
      been characterized. PRINCIPAL FINDINGS: Here we established new biochemical assay 
      conditions that enabled us to assess both polymerase activity with physiological 
      NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly 
      active RNA-dependent RNA polymerase activity at unbiased physiological NTP 
      substrate concentrations. With this robust enzyme activity, for the first time, 
      we were able to compare the enzyme fidelity of IAV Pol complex with that of 
      bacterial phage T7 RNA polymerase and the reverse transcriptases (RT) of human 
      immunodeficiency virus (HIV-1) and murine leukemia virus (MuLV), which are known 
      to be low and high fidelity enzymes, respectively. We observed that IAV Pol 
      displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and 
      equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex 
      showed increased fidelity at lower temperatures. Moreover, upon replacement of 
      Mg(++) with Mn(++), IAV Pol displayed increased polymerase activity, but with 
      significantly reduced processivity, and misincorporation was slightly elevated in 
      the presence of Mn(++). Finally, when the IAV nucleoprotein (NP) was included in 
      the reactions, the IAV Pol complex exhibited enhanced polymerase activity with 
      increased fidelity. SIGNIFICANCE: Our study indicates that IAV Pol is a high 
      fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be 
      important to counter-balance the multiple rounds of IAV genome amplification per 
      infection cycle, which provides IAV Pol with ample opportunities to generate and 
      amplify genomic founder mutations, and thus achieve optimal viral mutagenesis for 
      its evolution.
FAU - Aggarwal, Shilpa
AU  - Aggarwal S
AD  - Department of Microbiology and Immunology, University of Rochester Medical 
      Center, Rochester, New York, United States of America.
FAU - Bradel-Tretheway, Birgit
AU  - Bradel-Tretheway B
FAU - Takimoto, Toru
AU  - Takimoto T
FAU - Dewhurst, Stephen
AU  - Dewhurst S
FAU - Kim, Baek
AU  - Kim B
LA  - eng
GR  - HHSN266200700008C/AI/NIAID NIH HHS/United States
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20100429
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Viral Proteins)
RN  - EC 2.7.7.- (bacteriophage T7 RNA polymerase)
RN  - EC 2.7.7.49 (HIV Reverse Transcriptase)
RN  - EC 2.7.7.49 (RNA-Directed DNA Polymerase)
RN  - EC 2.7.7.6 (DNA-Directed RNA Polymerases)
SB  - IM
MH  - DNA-Directed RNA Polymerases/chemistry/*metabolism
MH  - HIV Reverse Transcriptase/chemistry/metabolism
MH  - Humans
MH  - Influenza A virus/*enzymology
MH  - Leukemia Virus, Murine/enzymology
MH  - RNA-Directed DNA Polymerase/chemistry/metabolism
MH  - Substrate Specificity
MH  - Viral Proteins/chemistry/metabolism
PMC - PMC2861597
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2010/05/11 06:00
MHDA- 2011/06/17 06:00
PMCR- 2010/04/29
CRDT- 2010/05/11 06:00
PHST- 2010/03/04 00:00 [received]
PHST- 2010/04/07 00:00 [accepted]
PHST- 2010/05/11 06:00 [entrez]
PHST- 2010/05/11 06:00 [pubmed]
PHST- 2011/06/17 06:00 [medline]
PHST- 2010/04/29 00:00 [pmc-release]
AID - 10-PONE-RA-16775R1 [pii]
AID - 10.1371/journal.pone.0010372 [doi]
PST - epublish
SO  - PLoS One. 2010 Apr 29;5(4):e10372. doi: 10.1371/journal.pone.0010372.