The influenza virus polymerase is a heterotrimer formed by the PB1, PB2 and PA subunits and is responsible for virus transcription and replication. We have expressed the virus polymerase complex by co-transfection of the subunit cDNAs, one of which was tandem affinity purification (TAP)-tagged, into human cells. The intracellular polymerase complexes were purified by the TAP approach, involving two affinity chromatography steps, IgG–Sepharose and calmodulin–agarose. Gel-filtration analysis indicated that, although most of the purified polymerase behaved as a heterotrimer, a significant proportion of the purified material migrated as polymerase dimers, trimers and higher oligomers. Co-purification of polymerase complexes alternatively tagged in the same subunit confirmed that the polymerase complex might form oligomers intracellularly. The implications of this observation for virus infection are discussed.
AreaE.,
Martín-BenitoJ.,
GastaminzaP.,
TorreiraE.,
ValpuestaJ. M.,
CarrascosaJ. L.,
OrtínJ.2004; Three-dimensional structure of the influenza virus RNA polymerase: localization of subunit domains. Proc Natl Acad Sci U S A 101:308–313[CrossRef]
BouloyM.,
PlotchS. J.,
KrugR. M.1978; Globin mRNAs are primers for the transcription of influenza viral RNA in vitro . Proc Natl Acad Sci U S A 75:4886–4890[CrossRef]
CevikB.,
SmallwoodS.,
MoyerS. A.2003; The L–L oligomerization domain resides at the very N-terminus of the sendai virus L RNA polymerase protein. Virology 313:525–536[CrossRef]
DengT.,
EngelhardtO. G.,
ThomasB.,
AkoulitchevA. V.,
BrownleeG. G.,
FodorE.2006; The role of Ran binding protein 5 (RanBP5) in the nuclear import and assembly of the influenza virus RNA polymerase complex. J Virol 80:11911–11919[CrossRef]
DetjenB. M.,
St AngeloC.,
KatzeM. G.,
KrugR. M.1987; The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex. J Virol 61:16–22
EltonD.,
DigardP.,
TileyL.,
OrtínJ.2005; Structure and function of the influenza virus RNP. In Current Topics in Influenza Virology pp 1–92Edited byKawaokaY.
Norfolk: Horizon Scientific Press;
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]
GastaminzaP.,
PeralesB.,
FalcónA. M.,
OrtínJ.2003; Influenza virus mutants in the N-terminal region of PB2 protein are affected in virus RNA replication but not transcription. J Virol 76:5098–5108
GonzálezS.,
OrtínJ.1999; Characterization of the influenza virus PB1 protein binding to vRNA: two separate regions of the protein contribute to the interaction domain. J Virol 73:631–637
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]
KlumppK.,
RuigrokR. W.,
BaudinF.1997; Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO J 16:1248–1257[CrossRef]
LeeM. T.,
BishopK.,
MedcalfL.,
EltonD.,
DigardP.,
TileyL.2002; Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase. Nucleic Acids Res 30:429–438[CrossRef]
MayerD.,
MolawiK.,
Martinez-SobridoL.,
GhanemA.,
ThomasS.,
BaginskyS.,
GrossmannJ.,
Garcia-SastreA.,
SchwemmleM.2007; Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches. J Proteome Res 6:672–682[CrossRef]
PaleseP.,
ShawM.2006; Orthomyxoviridae : the viruses and their replication. In Fields Virology , 5th edn. pp 1647–1689Edited byKnipeD. M.,
Howley.
Philadelphia: Lippincott Williams & Wilkins;
PoonL. L. M.,
PritloveD. C.,
FodorE.,
BrownleeG. G.1999; 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. J Virol 73:3473–3476
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]
RigautG.,
ShevchenkoA.,
RutzB.,
WilmM.,
MannM.,
SeraphinB.1999; A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032[CrossRef]
SmallwoodS.,
CevikB.,
MoyerS. A.2002; Intragenic complementation and oligomerization of the L subunit of the sendai virus RNA polymerase. Virology 304:235–245[CrossRef]
TileyL. S.,
HagenM.,
MathewsJ. T.,
KrystalM.1994; Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5′-end of the viral RNAs. J Virol 68:5108–5116
TorreiraE.,
SchoehnG.,
FernándezY.,
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]
VillacéP.,
MariónR. M.,
OrtínJ.2004; The composition of Staufen-containing RNA granules from human cells indicate a role in the regulated transport and translation of messenger RNAs. Nucleic Acids Res 32:2411–2420[CrossRef]
WangQ. M.,
HockmanM. A.,
StaschkeK.,
JohnsonR. B.,
CaseK. A.,
LuJ.,
ParsonsS.,
ZhangF.,
RathnachalamR.other authors2002; Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase. J Virol 76:3865–3872[CrossRef]