A reverse-genetics system for Influenza A virus using T7 RNA polymerase

Abstract

The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.

Molecular Determinants of Adaptation of Highly Pathogenic Avian Influenza H7N7 Viruses to Efficient Replication in the Human Host

“…To compare the polymerase activities of the CC and FC viruses, we used a T7 RNA polymerase promoter-based minigenome assay to study the effect of amino acid substitutions on polymerase activity in cells of mammalian and avian origin (18). The reporter minigenome consisted of the firefly luciferase open reading frame flanked by the NCRs of gene segment 8 of the CC virus.…”

“…This insert was cloned in negative sense orientation under the control of a bacteriophage T7 RNA polymerase promoter (18).…”

Interaction between Hantavirus Nucleocapsid Protein (N) and RNA-Dependent RNA Polymerase (RdRp) Mutants Reveals the Requirement of an N-RdRp Interaction for Viral RNA Synthesis

“…However, it is still a mystery how RdRp accesses the viral RNA (vRNA) genome, which is buried inside the compact nucleocapsid structure. Moreover, numerous reverse genetic systems have proven that RdRp from negative-strand RNA viruses requires assistance from N protein while performing its function inside the host cell (12)(13)(14)(15)(16). Based on these observations, we asked whether N protein directly interacts with RdRp and regulates its function.…”

Establishment of Canine RNA Polymerase I-Driven Reverse Genetics for Influenza A Virus: Its Application for H5N1 Vaccine Production

“…In MDCK cells, however, reverse genetics with the human PolI promoter does not work well, due to the host species specificity of the PolI promoter. Recently, another reverse genetics system with T7 RNA PolII was shown to support influenza virus generation in MDCK cells (2), although the efficiency of virus generation was inconsistent. In the present study, we established an alternative reverse genetics system driven by canine PolI and generated recommended H5N1 vaccine seed viruses in MDCK cells with high efficiency.…”