1. Determination of cold-adapted influenza virus (Orthomyxoviridae: Alphainfluenzavirus ) polymerase activity by the minigenome method with a fluorescent protein.
- Author
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Ivanov PA, Lyashko AV, Kost VY, Lomakina NF, Rtishchev AA, Bunkova NI, Timofeeva TA, Balanova MA, Ionov SA, Gorikov DV, and Markushin SG
- Subjects
- Viral Proteins genetics, Viral Proteins metabolism, Temperature, RNA-Dependent RNA Polymerase genetics, RNA-Dependent RNA Polymerase metabolism, Alphainfluenzavirus, Influenza A Virus, H1N1 Subtype genetics, Orthomyxoviridae genetics, Orthomyxoviridae metabolism
- Abstract
Introduction: Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier., Objective: The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method., Materials and Methods: Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct., Results: Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase., Conclusion: Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.
- Published
- 2023
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