The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit
Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30–42 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.
BrownleeG. G.,
SharpsJ. L.2002; The RNA polymerase of influenza A virus is stabilized by interaction with its viral RNA promoter. J Virol 76:7103–7113[CrossRef]
CarrS. M.,
CarneroE.,
Garcia-SastreA.,
BrownleeG. G.,
FodorE.2006; Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses. Virology 344:492–508[CrossRef]
ClaasE. C.,
OsterhausA. D.,
van BeekR.,
De JongJ. C.,
RimmelzwaanG. F.,
SenneD. A.,
KraussS.,
ShortridgeK. F.,
WebsterR. G.1998; Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351:472–477[CrossRef]
DeaneR.,
SchaferW.,
ZimmermannH. P.,
MuellerL.,
GorlichD.,
PrehnS.,
PonstinglH.,
BischoffF. R.1997; Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin- β but interacts differently with RanBP1. Mol Cell Biol 17:5087–5096
DengT.,
SharpsJ.,
FodorE.,
BrownleeG. G.2005; In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex. J Virol 79:8669–8674[CrossRef]
DengT.,
EngelhardtO. G.,
ThomasB.,
AkoulitchevA. V.,
BrownleeG. G.,
FodorE.2006; Role of Ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex. J Virol 80:11911–11919[CrossRef]
FodorE.,
SmithM.2004; The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex. J Virol 78:9144–9153[CrossRef]
FodorE.,
CrowM.,
MingayL. J.,
DengT.,
SharpsJ.,
FechterP.,
BrownleeG. G.2002; A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J Virol 76:8989–9001[CrossRef]
GabrielG.,
DauberB.,
WolffT.,
PlanzO.,
KlenkH. D.,
StechJ.2005; The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci U S A 102:18590–18595[CrossRef]
GabrielG.,
AbramM.,
KeinerB.,
WagnerR.,
KlenkH. D.,
StechJ.2007; Differential polymerase activity in avian and mammalian cells determines host range of influenza virus. J Virol 81:9601–9604[CrossRef]
GabrielG.,
HerwigA.,
KlenkH. D.2008; Interaction of polymerase subunit PB2 and NP with importin α 1 is a determinant of host range of influenza A virus. PLoS Pathog 4:e11[CrossRef]
GambottoA.,
Barratt-BoyesS. M.,
de JongM. D.,
NeumannG.,
KawaokaY.2008; Human infection with highly pathogenic H5N1 influenza virus. Lancet 371:1464–1475[CrossRef]
HaraK.,
SchmidtF. I.,
CrowM.,
BrownleeG. G.2006; Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding. J Virol 80:7789–7798[CrossRef]
HattaM.,
HattaY.,
KimJ. H.,
WatanabeS.,
ShinyaK.,
NguyenT.,
LienP. S.,
LeQ. M.,
KawaokaY.2007; Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog 3:e133[CrossRef]
HeimA.,
EbnetC.,
HarsteG.,
Pring-AkerblomP.2003; Rapid and quantitative detection of human adenovirus DNA by real-time PCR. J Med Virol 70:228–239[CrossRef]
KawaguchiA.,
NaitoT.,
NagataK.2005; Involvement of influenza virus PA subunit in assembly of functional RNA polymerase complexes. J Virol 79:732–744[CrossRef]
LabadieK.,
Dos Santos AfonsoE.,
Rameix-WeltiM. A.,
van der WerfS.,
NaffakhN.2007; Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells. Virology 362:271–282[CrossRef]
LambR. A.,
KrugR. M.2001; Orthomyxoviridae : the viruses and their replication. In Fields Virology , 4th edn. pp 1487–1531Edited byKnipeD. M.,
HowleyP. M.,
GriffinD. E.,
LambR. A.,
MartinM. A.,
RoizmanB.,
Strauss.
Philadelphia, PA: Lippincott, Williams and Wilkins;
LiM. L.,
RaoP.,
KrugR. M.2001; The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. EMBO J 20:2078–2086[CrossRef]
LiuM.,
HeS.,
WalkerD.,
ZhouN.,
PerezD. R.,
MoB.,
LiF.,
HuangX.,
WebsterR. G.,
WebbyR. J.2003; The influenza virus gene pool in a poultry market in South Central China. Virology 305:267–275[CrossRef]
MalkowskiM. G.,
QuartleyE.,
FriedmanA. E.,
BabulskiJ.,
KonY.,
WolfleyJ.,
SaidM.,
LuftJ. R.,
PhizickyE. M.other authors2007; Blocking S -adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing. Proc Natl Acad Sci U S A 104:6678–6683[CrossRef]
MassinP.,
van der WerfS.,
NaffakhN.2001; Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses. J Virol 75:5398–5404[CrossRef]
MurakamiY.,
NeromeK.,
YoshiokaY.,
MizunoS.,
OyaA.1988; Difference in growth behavior of human, swine, equine, and avian influenza viruses at a high temperature. Arch Virol 100:231–244[CrossRef]
MurphyB. R.,
SlyD. L.,
TierneyE. L.,
HosierN. T.,
MassicotJ. G.,
LondonW. T.,
ChanockR. M.,
WebsterR. G.,
HinshawV. S.1982b; Reassortant virus derived from avian and human influenza A viruses is attenuated and immunogenic in monkeys. Science 218:1330–1332[CrossRef]
NaffakhN.,
MassinP.,
EscriouN.,
Crescenzo-ChaigneB.,
van der WerfS.2000; Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses. J Gen Virol 81:1283–1291
NietoA.,
de la LunaS.,
BarcenaJ.,
PortelaA.,
ValcarcelJ.,
MeleroJ. A.,
OrtinJ.1992; Nuclear transport of influenza virus polymerase PA protein. Virus Res 24:65–75[CrossRef]
PeralesB.,
Sanz-EzquerroJ. J.,
GastaminzaP.,
OrtegaJ.,
SantarenJ. F.,
OrtinJ.,
NietoA.2000; The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis. J Virol 74:1307–1312[CrossRef]
PuigO.,
CasparyF.,
RigautG.,
RutzB.,
BouveretE.,
Bragado-NilssonE.,
WilmM.,
SeraphinB.2001; The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229[CrossRef]
ReganJ. F.,
LiangY.,
ParslowT. G.2006; Defective assembly of influenza A virus due to a mutation in the polymerase subunit PA. J Virol 80:252–261[CrossRef]
SalomonR.,
FranksJ.,
GovorkovaE. A.,
IlyushinaN. A.,
YenH. L.,
Hulse-PostD. J.,
HumberdJ.,
TrichetM.,
RehgJ. E.other authors2006; The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04. J Exp Med 203:689–697[CrossRef]
Sanz-EzquerroJ. J.,
de la LunaS.,
OrtinJ.,
NietoA.1995; Individual expression of influenza virus PA protein induces degradation of coexpressed proteins. J Virol 69:2420–2426
ShinyaK.,
HammS.,
HattaM.,
ItoH.,
ItoT.,
KawaokaY.2004; PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice. Virology 320:258–266[CrossRef]
SuzukiY.,
ItoT.,
SuzukiT.,
HollandR. E.Jr,
ChambersT. M.,
KisoM.,
IshidaH.,
KawaokaY.2000; Sialic acid species as a determinant of the host range of influenza A viruses. J Virol 74:11825–11831[CrossRef]
TaubenbergerJ. K.,
ReidA. H.,
LourensR. M.,
WangR.,
JinG.,
FanningT. G.2005; Characterization of the 1918 influenza virus polymerase genes. Nature 437:889–893[CrossRef]
TorreiraE.,
SchoehnG.,
FernandezY.,
JorbaN.,
RuigrokR. W.,
CusackS.,
OrtinJ.,
LlorcaO.2007; Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer. Nucleic Acids Res 35:3774–3783[CrossRef]
The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit