Der Einfluss von N6-Methyladenosin auf die Replikation von Influenzaviren

RNA from different organisms contains more than 100 different chemically modified nucleosides, but their functions are largely unknown. N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA and is post-transcriptionally installed by a methyltransferase complex consisting of METTL3, METTL14 and WTAP within the consensus motif [G/A/T][G>A]m6AC[T>A>C]. Therefore, the sequence motif GGACT is the most common m6A motif. M6A is bound by proteins from the YTH and hnRNP protein family, which function as reader proteins and regulate cellular processes like translation, splicing and RNA decay. Interestingly, viral mRNA has also been reported to contain m6A but the role for virus replication and pathogenicity still has to be elucidated. Studies from the 1970s indicate that influenza virus mRNA also contains m6A but the exact positions of m6A have not yet been determined. In this thesis, the influence of m6A on the replication of the influenza strain A/Puerto Rico/8/1934 H1N1 was analyzed. For this purpose, recombinant viruses were generated which contain mutations in the m6A consensus motifs GGACT of the viral NS, NA or HA mRNAs. These mutations will abrogate the N6-methylation at the respective position without inducing a change in the amino acid sequence of the protein. The viruses were characterized by replication experiments and analysis of the viral RNA and protein expression, and then compared to the wt virus. It could be shown that the virus with the mutated GGACT motifs in the HA mRNA (HAmut1-4) replicated less efficient when compared to the wt virus and showed a reduced HA mRNA and HA protein expression. In contrast, the mutations in the GGACT motifs of the NS and NA mRNA did not affect viral replication. To link the observations from the replication studies to the methylation status of the viruses, the m6A positions in the mRNA from the wt virus and from the virus HAmut1-4 were determined by the method of methylated RNA immunoprecipitation with next generation sequencing (MeRIP-Seq). This analysis showed that the GGACT motifs in the NS and NA mRNA of the wt virus were not methylated and therefore confirmed the results from the replication studies, in which the viruses with mutations at these sites replicated normally. In the HA mRNA, only one of the four GGACT sites was methylated. The mRNA from the wt virus contained a methylation at the second GGACT motif, which was abrogated in the mRNA from the virus HAmut1-4 with the mutated GGACT motifs. In further experiments recombinant influenza viruses were generated in which the GGACT motifs in the HA mRNA were mutated individually. Replication studies with these viruses could show that the mutation of the second GGACT motif lead to a reduced replication efficiency and to a decreased expression of HA mRNA and HA protein. Therefore, the reduced replication efficiency of the virus HAmut1-4 with four mutated GGACT motifs in the HA-mRNA could be linked solely to the single mutation at the second GGACT motif and to a resulting loss of m6A at this position. The MeRIP-Seq could determine further m6A modifications in the NA, M, NA, NP and HA mRNA, especially in the 5’ and 3’ regions. Further studies have to elucidate the functions of these m6A modifications. The detection of an influenza-mediated regulation of the m6A-associated enzymes should reveal how m6A regulates viral mRNA and protein expression during an influenza infection. In this thesis, however, an influenza-mediated change in the mRNA expression of different methyltransferases, demethylases and reader proteins could not be shown. With regard to different studies, which indicate a role of YTHDF2 in the m6A-mediated increase of protein translation, the YTHDF2 protein expression was also analyzed during an influenza infection in human cell lines. However, the virus infection did not induce any visible change in the YTHDF2 protein expression. Therefore, further studies have to reveal how m6A influences the replication and protein expression of influenza viruses. In summary, this thesis could show that the loss of an m6A modification in the viral mRNA lead to a reduced replication efficiency of influenza virus. These data suggest that the inhibition of m6A modifications could have the potential to inhibit influenza replication and could therefore be used as a therapy against influenza infection.