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Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

ABSTRACT: An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells.

SUBMITTER: Lu G

PROVIDER: S-EPMC4926170 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

Lu Gang G He Dong D Wang Zengchao Z Ou Shudan S Yuan Rong R Li Shoujun S

Viruses 20160531 6

An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our stud ...[more]

PMID: 27258298

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Project description:Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.

Project description:BackgroundRecent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells.ResultsThe eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 106 to 107 plaque forming units per ml by 5 to 7 days post-transfection.ConclusionThis rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.

Dataset Information

Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

Publications

Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

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