Recently, we developed a system for the generation of influenza A virus by cotransfecting only eight plasmids from which negative-sense vRNA and positive-sense mRNA are expressed (Hoffmann et al., Proceedings of the National Academy of Sciences, USA 97, 6108–6113, 2000). Here we report the establishment of a different transcription system for the expression of virus-like RNAs, allowing the intracellular synthesis of noncapped positive-sense cRNA and 5′-capped mRNA from one template. Cotransfection of eight RNA pol I–pol II tandem promoter plasmids containing the cDNA of A/WSN/33 (H1N1) resulted in the generation of infectious influenza A virus, albeit with a lower yield than the bidirectional system. Our approach of producing either vRNA and mRNA or cRNA and mRNA intracellularly from a minimum set of plasmids should be useful for the establishment or optimization of reverse genetics systems for other RNA viruses.
AlmazánF.,
GonzálezJ. M.,
PénzesZ.,
IzetaA.,
CalvoE.,
Plana-DuránJ.,
EnjuanesL.2000; Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proceedings of the National Academy of Sciences, USA 97:5516–5521
BridgenA.,
ElliottR. M.1996; Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proceedings of the National Academy of Sciences, USA 93:15400–15404
EnamiM.,
LuytjesW.,
KrystalM.,
PaleseP.1990; Introduction of site-specific mutations into the genome of influenza virus. Proceedings of the National Academy of Sciences USA: 87:3802–3805
FodorE.,
DevenishL.,
EngelhardtO. G.,
PaleseP.,
BrownleeG. G.,
García-SastreA.1999; Rescue of influenza A virus from recombinant DNA. Journal of Virology 73:9679–9682
GonzálezS.,
OrtínJ.1999b; Characterization of the influenza virus PB1 protein binding to vRNA: two separate regions of the protein contribute to the interaction domain. Journal of Virology 73:631–637
GonzálezS.,
ZürcherT.,
OrtínJ.1996; Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure. Nucleic Acids Research 24:4456–4463
HoffmannE.,
NeumannG.,
HobomG.,
WebsterR. G.,
KawaokaY.2000a; ‘Ambisense’ approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template. Virology 267:310–317
HoffmannE.,
NeumannG.,
KawaokaY.,
HobomG.,
WebsterR. G.2000b; A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences, USA 97:6108–6113
HsuM. T.,
ParvinJ. D.,
GuptaS.,
KrystalM.,
PaleseP.1987; Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle. Proceedings of the National Academy of Sciences, USA 84:8140–8144
LachmannR. H.,
BrownC.,
EfstathiouS.1996; A murine RNA polymerase I promoter inserted into the herpes simplex virus type 1 genome is functional during lytic, but not latent, infection. Journal of General Virology 77:2575–2582
LambR. A.,
KrugR. M.1996; Orthomyxoviridae . The viruses and their replication. In Fields Virology pp 1353–1395 Edited by
FieldsB. N.,
KnipeD. M.,
HowleyP. M.
Philadelphia: Lippincott–Raven;
LeeK. J.,
NovellaI. S.,
TengM. N.,
OldstoneM. B. A.,
de la TorreJ. C.2000; NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs. Journal of Virology 74:3470–3477
LiM. L.,
RamirezB. C.,
KrugR. M.1998; RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites. EMBO Journal 17:5844–5852
LuytjesW.,
KrystalM.,
EnamiM.,
ParvinJ. D.,
PaleseP.1989; Amplification, expression, and packaging of a foreign gene by influenza virus. Cell 59:1107–1113
NeumannG.,
WatanabeT.,
ItoH.,
WatanabeS.,
GotoH.,
GaoP.,
HughesM.,
PerezD. R.,
DonisR.,
HoffmannE.,
HobomG.,
KawaokaY.1999; Generation of influenza A viruses entirely from cloned cDNAs. Proceedings of the National Academy of Sciences, USA 96:9345–9350
PalmerT. D.,
MillerA. D.,
ReederR. H.,
McStayB.1993; Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter. Nucleic Acids Research 21:3451–3457
PleschkaS.,
JaskunasR.,
EngelhardtO. G.,
ZürcherT.,
PaleseP.,
García-SastreA.1996; A plasmid-based reverse genetics system for influenza A virus. Journal of Virology 70:4188–4192
RobertsA.,
RoseJ. K.1998; Recovery of negative-strand RNA viruses from plasmid DNAs: a positive approach revitalizes a negative field. Virology 247:1–6
ToyodaT.,
AdyshevD. M.,
KobayashiM.,
IwataA.,
IshihamaA.1996; Molecular assembly of the influenza virus RNA polymerase: determination of the subunit-subunit contact sites. Journal of General Virology 77:2149–2157
UlmanenI.,
BroniB. A.,
KrugR. M.1981; Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription. Proceedings of the National Academy of Sciences, USA 78:7355–7359