Unidirectional RNA polymerase I–polymerase II transcription system for the generation of influenza A virus from eight plasmids

2000/12/01 by Erich Hoffmann, Robert G. Webster · 26 citations
Medicine · #Bacteriophage #Biology #Complementary DNA #Gene #Genetics #Influenza A virus #Influenza Virus Research Studies #Messenger RNA #Molecular biology #Plasmid #Polymerase #RNA #RNA polymerase #Respiratory viral infections research #Reverse transcriptase #T7 RNA polymerase #Transcription (linguistics) #Viral Infections and Immunology Research #Virology #Virus

paper · doi:10.1099/0022-1317-81-12-2843

openalex publication_date 2000/12/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/08/01

Abstract

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.

Cited by

• Evolution of H5N1 Avian Influenza Viruses in Asia
• Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. [europepmc]
• Viral genetic determinants of H5N1 influenza viruses that contribute to cytokine dysregulation. [europepmc]
• Contribution of vaccine-induced immunity toward either the HA or the NA component of influenza viruses limits secondary bacterial complications. [europepmc]
• NS-based live attenuated H1N1 pandemic vaccines protect mice and ferrets. [europepmc]
• In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. [europepmc]
• Host Protein Moloney Leukemia Virus 10 (MOV10) Acts as a Restriction Factor of Influenza A Virus by Inhibiting the Nuclear Import of the Viral Nucleoprotein. [europepmc]
• Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains. [europepmc]
• Viral RNA Degradation and Diffusion Act as a Bottleneck for the Influenza A Virus Infection Efficiency. [europepmc]
• HIST1H1C Regulates Interferon-β and Inhibits Influenza Virus Replication by Interacting with IRF3. [europepmc]
• Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses. [europepmc]
• Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice. [europepmc]
• A Novel Role for PDZ-Binding Motif of Influenza A Virus Nonstructural Protein 1 in Regulation of Host Susceptibility to Postinfluenza Bacterial Superinfections. [europepmc]
• Novel Role for miR-1290 in Host Species Specificity of Influenza A Virus. [europepmc]
• Naturally occurring mutations in PB1 affect influenza A virus replication fidelity, virulence, and adaptability. [europepmc]
• Rapid Generation of Attenuated Infectious Bursal Disease Virus from Dual-Promoter Plasmids by Reduction of Viral Ribonucleoprotein Activity. [europepmc]
• Influenza Reverse Genetics-Historical Perspective. [europepmc]
• Truncation or Deglycosylation of the Neuraminidase Stalk Enhances the Pathogenicity of the H5N1 Subtype Avian Influenza Virus in Mallard Ducks. [europepmc]
• Interaction of NEP with G Protein Pathway Suppressor 2 Facilitates Influenza A Virus Replication by Weakening the Inhibition of GPS2 to RNA Synthesis and Ribonucleoprotein Assembly. [europepmc]
• A Single-Cycle Influenza A Virus-Based SARS-CoV-2 Vaccine Elicits Potent Immune Responses in a Mouse Model. [europepmc]
• Active Components of Commonly Prescribed Medicines Affect Influenza A Virus-Host Cell Interaction: A Pilot Study. [europepmc]
• Generation of a pdmH1N1 2018 Influenza A Reporter Virus Carrying a mCherry Fluorescent Protein in the PA Segment. [europepmc]
• Comparison of CpG- and UpA-mediated restriction of RNA virus replication in mammalian and avian cells and investigation of potential ZAP-mediated shaping of host transcriptome compositions. [europepmc]
• Genetic characterization and pathogenicity of a Eurasian avian-like H1N1 swine influenza reassortant virus. [europepmc]
• Viral vectored vaccines: design, development, preventive and therapeutic applications in human diseases. [europepmc]
• Preclinical Evaluation of TB/FLU-04L-An Intranasal Influenza Vector-Based Boost Vaccine against Tuberculosis. [europepmc]

Related

• Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection
• Adaptation differences and mechanisms of influenza viruses to ANP32 proteins across species
• Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus
• Amplification of RNA by an RNA polymerase ribozyme
• First Versatile Reverse Genetics System for DNA Viruses Using Circular Polymerase Extension Reaction