Genotype-dependent and non-gradient patterns of RSV gene expression

Abstract

Respiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3 promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.\n\nAuthor SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3 promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.