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

Abstract

30Respiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA 31 virus and a leading cause of severe lower respiratory tract illness in infants and the 32 elderly. Transcription of the ten RSV genes proceeds sequentially from the 3' promoter 33 and requires conserved gene start (GS) and gene end (GE) signals. Previous studies 34 using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of 35 gene transcription. However, recent reports show data that appear inconsistent with a 36 gradient. To better understand RSV transcriptional regulation, mRNA abundances from 37 five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines 38 and cotton rats infected with virus isolates belonging to four different genotypes (GA1, 39 ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours 40 post-infection. Steady-state patterns were genotype-specific and non-gradient, where 41 mRNA levels from the G (attachment) gene exceeded those from the more promoter-42 proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account 43 for the non-gradient patterns observed, indicating that relative mRNA levels more 44 strongly reflect transcription than decay. While the GS signal sequences were highly 45 conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, 46 was variable, suggesting polymerase recognition of GS signal conformation affects 47 transcription initiation. The effect of GS N-phase on transcription efficiency was tested 48 using dicistronic minigenomes. Ratios of minigenome gene expression showed a 49 switch-like dependence on N-phase with a period of seven nucleotides. Our results 50 indicate that RSV gene expression is in part sculpted by polymerases that initiate 51 transcription with a probability dependent on GS signal N-phase. 3 52 Author Summary 53 54RSV is a major viral pathogen that causes significant morbidity and mortality, 55 especially in young children. Shortly after RSV enters a host cell, transcription from its 56 nonsegmented negative-strand (NNS) RNA genome starts at the 3' promoter and 57 proceeds sequentially. Transcriptional attenuation is thought to occur at each gene 58 junction, resulting in a gradient of gene expression. However, recent studies showing 59 non-gradient levels of RSV mRNA suggest that transcriptional regulation may have 60 additional mechanisms. We show using RSV isolates belonging to four different 61 genotypes that gene expression is genotype-dependent and one gene (the G or 62 attachment gene) is consistently more highly expressed than an upstream neighbor. We 63 hypothesize that variable alignment of highly conserved gene start (GS) signals with 64 nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-65 gradient patterns of gene expression. We show using dicistronic RSV minigenomes 66 wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase 67 affects gene expression....

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus

“…and genotype-dependent transcription in RSV and Ebola (22,23). During transcription, using a single promoter in the 3' leader region (Le) of the genome, the polymerase initiates and terminates the mRNA transcription in response to the gene start (GS) and gene end (GE) signals.…”

Structures of the Mononegavirales Polymerases

“…Typically, the RdRP produces a gradient level of viral mRNAs with the attenuation of the downstream mRNAs at each gene junction (71,72). Recent studies showed the non-gradient and genotype-dependent transcription in HRSV and EBOV, suggesting alternative gene expression strategies (73,74) (Fig. 1, lower part).…”

Cryo-Electron Microscopy Structures of the Pneumoviridae Polymerases

“…A cis-acting sequence gene end (GE) signals polyadenylation and termination; (iii) the polymerase stays on the template, reinitiates and caps the downstream mRNAs, and terminates and polyadenylates the upstream mRNAs in response to GS and GE signals of downstream genes (26,27); (iv) Like other Mononegavirales, the attenuation of the downstream mRNA occurs at each gene junction for most genes (25). Recent studies indicated the existence of nongradient and genotypedependent transcription in RSV, suggesting the possibility of such nongradient gene expression in other Mononegavirales (34,36). Through this process of sequential attenuation of transcription, L produces ten viral mRNAs with appropriate ratios (32) (Fig.…”