The untranslated regions within viral segments are the essential promoter elements required for the initiation of viral replication and transcription. The end of the UTR sequence and part of the ORF sequence constitute the packaging signal for progeny viruses. To explore the influence of single-point and multi-site joint mutations in the UTR of the NA gene on the viral expression, we select clones with upregulated expression of the reporter gene and analyze their sequence characteristics. Bioinformatics methods were used to analyze polymorphisms in the untranslated region (UTR) of the neuraminidase gene of the H9N2 influenza A virus. Using the RNA polymerase I reporting system with enhanced green fluorescence protein (EGFP) gene as the reporter gene, libraries containing random mutations at sites within the N2 UTR were constructed using random mutagenesis. The mutants were selected from the randomized mutagenesis libraries for the N2-UTR. The N2-UTR-RNA polymerase I fluorescence reporter system was identified by sequencing and transfected into infected MDCK cells. The expression of the reporter EGFP was observed using fluorescence microscopy, and the relative fluorescence intensity was measured using a multifunctional microplate reader to analyze the expression of the reporter gene (EGFP) qualitatively and quantitatively. Herein, an RNA polymerase reporter system was constructed to rescue the mutated viruses and measure their tissue culture infective dose (TCID50). The results showed that the U13 → C13 mutation in the 3′end of the NA gene promoted the expression of viral RNA and protein, and mutation of other sites within the UTR could differentially regulate viral genomic transcription and translation. These data showed that the U13 → C13 mutation within the variable region of the 3′UTR of the NA gene in the H9N2 influenza virus promotes viral genomic expression and infection.
H9N2 influenza A virus The variable region EGFP N2-UTR RNA polymerase I fluorescence reporter system
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This work was supported by the National Natural Science Foundation of China (81871631), the Shenzhen Special Fund Project for Strategic Emerging Industry Development (JCYJ20170306160244540), the Shenzhen Research Project for the Health and Family Planning System (201602002), and the Guangdong Provincial Medical Science and Technology Foundation (A2018269).
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflicts of interest to disclose.
This article does not contain any studies with human participants or animals that were performed by any of the authors.
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