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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....

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host.

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