Study of novel splicing variants and splicing regulation mechanism of the M segment of influenza A viruses

Abstract

The M segment of influenza A viruses (IAVs) is known to generate four mRNA transcripts (M1, M2, mRNA3, and mRNA4) through alternative splicing. It is postulated that other novel M spliced forms may exist that facilitate viral functions. Avian H9N2 viruses are the primary donor of internal genes to reassortant H5 and H7 viruses that have caused human infections and other subtypes of avian influenza viruses with zoonotic potential. Despite significant research in this area, the molecular mechanisms of H9N2 virus replication remain elusive. Abnormal splicing of the M segment has been reported to affect virus replication, therefore, apart from finding novel splicing variants of the M segment, it is also important to identify factors that influence M splicing. In the present study, I applied reverse transcription PCR targeting viral M mRNAs of the H9N2 virus to look for new mRNA splicing variants. A novel mRNA, designated M6, was found and M6 protein was detectable through immunoprecipitation and western blotting. M6 protein colocalized with M2 protein in the cytoplasm of H9N2-infected cells. To check whether M6 is present in the virion, I purified viruses by ultracentrifugation and examined by silver staining and mass spectrometry. The results showed that M6 was not incorporated in the H9N2 virion. In addition, several posttranslational modifications were found in M1 that correlate with previously reported adaptive mutations in the H9N2 virus, confirming the importance of posttranslational modification of viral proteins in the IAV adaptation process. I found that M6 expression conferred a low-level growth advantage to the H9N2 virus in both mammalian and avian cells, influencing both single-cycle replication and competitiveness. Furthermore, my study indicated that the M6 motif emerged in early 1999 and became more predominant in 2014, supporting the importance of M6 existence during the process of H9N2 virus evolution. Regarding the major products expressed from the M segment, the M2/M1 mRNA splicing ratio varies among different IAV strains. To understand this phenomenon, I focused on several cis-acting elements and trans-acting factors. For cis-acting factors, my study found that nucleotide 18 and nucleotide 21, which are positioned within the segment-specific noncoding region of the M segment, vary among IAV strains and could affect M splicing. For trans-acting elements, my results demonstrated that the nuclear localization of NS1 and the expression level of NEP are essential for proper M splicing. Besides viral proteins, I found that the cellular protein YTHDC1 could affect M splicing and consequently IAV replication. Furthermore, NS1 binding the ability of NS1 to bind to M mRNA was affected by M-A18G substitution, indicating that cis-acting factors and trans-acting elements can combine to impact M splicing. In conclusion, my project revealed a novel viral splicing variant in H9N2 virus infected cells and investigated cis-acting factors and trans-acting elements that influence M splicing of various IAV strains. These findings concerning M splicing variants and their roles in host adaptation will help us understand the evolution of IAVs and provide new insights into the regulation of virus replication through the viral spliceosome.