| Human respiratory syncytial virus (HRSV) is the leading viral cause of severe lower respiratory tract disease in young children. A member of the order Mononegavirales, HRSV has a single-stranded, negative-sense RNA genome. Transcription of the viral genome proceeds sequentially from a single 3' transcriptional promoter, and attenuation at each gene junction creates a gradient of transcription. Therefore, gene expression of the Mononegavirales is primarily controlled at the transcriptional level. However, unlike several other viruses in this order, the sequences, termination efficiencies, and attenuation levels vary among the HRSV gene junctions, which could potentially have significant effects on downstream gene expression. The work presented in this dissertation examines the role of gene end and intergenic region sequence variation in transcriptional control and virus replication. We determined the gene start, gene end, and intergenic region sequences of each of the 10 HRSV genes from 14 clinical isolates and compared the isolate sequences to those present in the prototype A2 strain. While no changes were found in any of the gene start sequences, the gene end and intergenic region sequences contained variations from the A2 sequence. When we examined the potential transcriptional effects of the clinical isolate sequences, we found that certain variations significantly affected termination and downstream mRNA synthesis. We then investigated whether the variation in transcriptional control sequences found in the different genes is a key factor for optimal virus replication by engineering a recombinant HRSV containing identical gene end and intergenic region sequences at each of its first eight gene junctions. Recovery and subsequent analyses indicated that this virus was debilitated. Upon passage of the recovered virus in cell culture, a virus population with an improved replication phenotype was isolated. This virus population contained changes from the engineered sequence at three gene ends. This indicated that varied gene junction sequences are required for wild-type levels of HRSV growth, possibly due to a requirement for varied transcriptional control signals at the different gene junctions. Together, these data suggest that the varied gene junction sequences play a crucial role in the life cycle of HRSV. |
