Gene expression regulation in pneumoviruses

Members of the Pneumoviridae virus family are responsible for severe respiratory tract disease in their hosts. Human respiratory syncytial virus (hRSV) is responsible for over 200,000 deaths worldwide each year and bovine respiratory syncytial virus (bRSV) causes major economic loss to the cattle industry worldwide. The current model for all nonsegmented negative-sense single stranded RNA virus gene expression, is that mRNA is generated in a polar gradient, with decreasing levels of mRNA transcribed from genes further along the genome from the 3´ end. With the exception of translation of ORF-2 located on the bicistronic M2 mRNA, translation of Pneumoviridae mRNAs is thought to be regulated through the levels of mRNA abundance. Translation of M2 ORF-2 has been characterised as being regulated by the non-canonical mechanism of coupled translation termination/initiation in pneumonia virus of mice (PVM), hRSV and avian metapneumovirus (APV). This mechanism is reliant on a proportion of the elongating ribosome translating the upstream M2 ORF-1, terminating and reinitiating translation of M2 ORF-2. Although the initiation site for M2 ORF-2 is similar in bRSV to other members of this family that use the mechanism of coupled translation, the mechanism has not been characterised. Using the technique of ribosomal profiling to analyse steady state viral mRNA abundance and viral translation in both hRSV and bRSV-infected cells, it was observed that for certain viral mRNAs, levels of mRNA abundance did not follow the standard polar transcription model. This was characterised by an increase in the levels of mRNA abundance between the mRNA’s respective gene and its upstream neighbour. The increase was observed in the same group of mRNAs in both viruses suggesting that factors other than the transcription polar gradient influence levels of viral mRNA abundance. It was also observed that levels of proportional translation did not match the respective proportional levels of mRNA abundance for certain viral mRNAs in both viruses. This would suggest that translation of viral genomes is not primarily controlled by mRNA abundance and instead other translational regulatory factors influence levels of translation. The mechanism of bRSV M2 ORF-2 translation was also characterised using reporter plasmids assays. It was identified that the mechanism of initiation of translation of M2 ORF2 used, was not that of coupled translation termination/initiation used by other members of this family. Instead it was observed that translation of M2 ORF-2 used an internal initiation mechanism located inside M2 ORF-1 to initiate translation. The mechanism of coupled translation termination/initiation used for translation of PVM M2 ORF-2 was also further characterised. It was observed that translation of M2 ORF-2 was reliant on upstream sequence in the M2 ORF-1 sequence. A predicted mRNA secondary structure was identified in this region and when disrupted, inhibited translation of M2 ORF2. This was similar to the mechanism of coupled translation used in hRSV, suggesting that the mechanism used by this family is reliant on a mRNA secondary structure located upstream of the initiation site.