Structural Analysis:Genome/Respiratory Syncytial Virus

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Abstract

Respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral agent of pediatric respiratory tract disease worldwide. It currently lacks a vaccine or effective antiviral therapy. RSV is in Family Paramyxoviridae of the nonsegmented negative strand RNA viruses or mononegaviruses, and is the most complex member of the group. We previously determined the complete sequence of RSV genomic RNA and mapped its genes. This included identification of transcription signals that begin and end each gene, the gene-start and gene-end signals, respectively, as well as the intergenic regions of 1 to 56 nucleotides in length that lie between the genes. Mutational analysis of the gene-end signal was performed to determine whether the naturally-occurring variation in this semi-conserved sequence was of functional significance. This showed that the sequence differences indeed affect the efficiency of termination and polyadenylation. However, when these changes were introduced into infectious recombinant virus, they had little or no effect on virus replication in vitro or in the respiratory tract of mice, indicating that these variable nucleotides likely do not represent important inter-strain differences. Work is also in progress to map and characterize the major viral promoters that lie at the 3' end of the genome and its encoded replicative intermediate or antigenome. Ongoing deletional analysis of minireplicons and complete recombinant virus indicates that most or all of each promoter lies within the first ~50 nucleotides of each end. We further characterized the genomic promoter by mutational analysis of individual residues, and showed that the core of the promoter consists of the first 11 positions from the end. Mutations at certain positions affected RNA replication and transcription equally, while at other positions RNA replication alone was affected. This indicated that the promoter requirements of the two processes overlapped but were not identical. Furthermore, transcription, but not replication, required that the first gene-start signal be spaced correctly relative to the 3'end, implying that this might serve as part of the transcription promoter. The intergenic regions do not seem to contain any important cis-acting signals, and indeed the intergenic region of a dicistronic minigenome could be deleted altogether without a detectable effect on RNA synthesis and sequential transcription. In complete recombinant virus, deletion within an intergenic sequence, or the insertion of an unnaturally-long intergenic region, had very little effect on viral gene expression or growth, supporting the idea that these are relatively unimportant spacer regions.