tetano
Editor, Senior Moderator
RNA Biol. 2017 Jun 29:0. doi: 10.1080/15476286.2017.1338243. [Epub ahead of print]
[h=1]A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.[/h] Spronken MI[SUP]1[/SUP], van de Sandt CE[SUP]1[/SUP], de Jongh EP[SUP]1,[/SUP][SUP]2[/SUP], Vuong O[SUP]1[/SUP], van der Vliet S[SUP]1[/SUP], Bestebroer TM[SUP]1[/SUP], Olsthoorn RCL[SUP]3[/SUP], Rimmelzwaan GF[SUP]1[/SUP], Fouchier RAM[SUP]1[/SUP], Gultyaev AP[SUP]1,[/SUP][SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared to the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.
PMID: 28662365 DOI: 10.1080/15476286.2017.1338243
[h=1]A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.[/h] Spronken MI[SUP]1[/SUP], van de Sandt CE[SUP]1[/SUP], de Jongh EP[SUP]1,[/SUP][SUP]2[/SUP], Vuong O[SUP]1[/SUP], van der Vliet S[SUP]1[/SUP], Bestebroer TM[SUP]1[/SUP], Olsthoorn RCL[SUP]3[/SUP], Rimmelzwaan GF[SUP]1[/SUP], Fouchier RAM[SUP]1[/SUP], Gultyaev AP[SUP]1,[/SUP][SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared to the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.
PMID: 28662365 DOI: 10.1080/15476286.2017.1338243