Variations in transcription termination signals of human respiratory syncytial virus clinical isolates affect gene expression

Abstract

Human respiratory syncytial virus (HRSV) has a single-stranded, negative-sense RNA genome with 10 genes encoding 11 proteins. Sequences at the beginning of the HRSV genes are highly conserved; however, the gene end sequences vary around a semiconserved consensus sequence, and the nontranscribed intergenic regions vary in both length and sequence. The regions at the junctions between HRSV genes (the gene end sequence of an upstream gene, intergenic region, and the gene start sequence of a downstream gene) contain elements required for efficient termination of the upstream gene and transcription of the downstream gene. Previous studies have examined variation in the HRSV coding sequences, but none have systematically analyzed the noncoding transcriptional control regions for variability. We determined the gene start and gene end sequences of each of the 10 HRSV genes from 14 clinical isolates for variations from the sequence of the prototype A2 strain. No changes were found in any of the gene start sequences. Eight of the 10 gene end sequences, however, contained variations. Several of these, a U(4)-tract instead of a U(6)- or U(5)-tract at the M and SH gene ends, respectively, (U(4)A) and an A-to-G change at position four in the G gene end (A4G), were predicted to affect termination and were examined for their effects on transcription. The changes were found to inhibit transcriptional termination, resulting in increased polycistronic readthrough and correspondingly reduced initiation of the downstream monocistronic mRNA. Viruses with the A4G variant G gene end sequence produced less F protein than those with A2-like G gene end sequences. Examination of additional G gene end sequences available in GenBank revealed that the observed A4G variation was restricted to one phylogenetic lineage of HRSV. All viruses examined within this lineage possessed this variant G gene end sequence. The data presented show that the gene end sequences of naturally occurring HRSV clinical isolates vary from those of the prototypic A2 strain and that certain of these changes inhibit efficient transcriptional termination and downstream gene expression.

Direct whole-genome deep-sequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter- and intra-host evolution

“…Other previously explored drug targets within the RSV genome include highly conserved regions of the N and F proteins (ALN-RSV01/RSV-604 and motavizumab, respectively) (Dormitzer et al , 2012; Empey et al , 2010). Regions containing signals that direct viral mRNA transcription or antigenome synthesis, such as the 3′ extragenic leader and the gene start and gene end sequences, were also found to be conserved in our study () (Collins et al , 1991; Fearns et al , 2000; Kuo et al , 1996, 1997; Mink et al , 1991; Moudy et al , 2003).…”

Whole genome characterization of non-tissue culture adapted HRSV strains in severely infected children

“…In four clinical strains nucleotide substitutions have been observed at five different positions in M2-L gene overlap sequence, while changes at only two of positions were reported earlier by Moudy et al [59]. Moreover, nucleotide change A-26-G observed in RSV-12 and RSV-13 led to changes of amino acid N-6-S in L protein.…”

Whole Genome Sequencing and Evolutionary Analysis of Human Respiratory Syncytial Virus A and B from Milwaukee, WI 1998-2010

“…. This in turn leads to decreased protein produced for genes at the 5′ end of the genome [36], [56]. It is in this manner that the termination efficiency of the GE seems to be used by RSV to regulate the production of its proteins.…”

“…It is in this manner that the termination efficiency of the GE seems to be used by RSV to regulate the production of its proteins. Multiple studies have shown that the transcription termination efficiency is principally regulated by the specific GE sequence with a little influence from individual bases of the 3′NCR and IGS of a gene [36]–[38], [40], [56]. The GE of the RSV A SH gene has been described as the ideal termination sequence and its high termination efficiency is evidenced by the lack of detectable SH-G dicistronic mRNA in cell culture.…”