Understanding poxvirus gene expression could lead to better vaccine vectors

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Cascade regulation of vaccinia virus gene expression is modulated by multistage promoters

Poxviruses are DNA viruses noted for their ability to replicate in the cytoplasm, their medical importance and their usefulness as vaccine vectors. Replication outside the nucleus relies on poxvirus-encoded proteins for genome replication and transcription. Transcription mapping of the genome of vaccinia virus, the prototype poxvirus, revealed 118 genes transcribed before genome replication and 93 only afterwards. Post-replicative gene expression is subdivided into intermediate and late classes that are regulated by stage-specific transcription factors. Fifty-three of the post-replicative genes are classified as intermediate because they are expressed in the absence of late transcription factors; the remaining post-replicative genes are classified as late.

The purpose of our study was to determine whether any of the intermediate genes have dual promoters that can be recognized by late as well as intermediate transcription factors. We devised a specific assay by infecting a cell line that stably expresses the late transcription factors with a mutant vaccinia virus that expresses RNA polymerase but neither intermediate nor late transcription factors. Each intermediate promoter was individually transfected and tested for late promoter activity using a luciferase read out.

 

The activities varied from none detectable to values approaching that of a strong late promoter. Consensus promoter sequences for early, strict intermediate, dual intermediate/late and late promoters are shown in the figure below. Intermediate promoter activity correlated with a T followed by As at the RNA start site and upstream As centered around nucleotide -15; late promoter activity correlated with a TAAAT at the start site and upstream Ts centered around nucleotide -11. Intermediate/late promoters had features of both strict intermediate and late promoters. Sequences that correlated with intermediate and late transcription were confirmed by mutagenesis. Multistage promoters enable poxviruses to refine the level and duration of expression of individual genes.

 

This new knowledge can be applied to the development of improved vaccine vectors. Increased amounts of immunogenic proteins can be expressed by employing dual promoters that are active at both intermediate and late times after vaccinia virus infection.

 

Consensus promoter sequences. Motif logos of vaccinia virus promoters were generated by the program MEME. Motifs representing early and late promoters were previously described (Yang, Z., Reynolds, S. E., Martens, C. A., Bruno, D. P., Porcella, S. F., and Moss, B., 2011. J. Virol. 85, 9899-9908). Intermediate and intermediate/late promoter logos were derived from 10 intermediate promoters with negligible late activities and 10 promoters with the highest late activities, respectively.
Consensus promoter sequences. Motif logos of vaccinia virus promoters were generated by the program MEME. Motifs representing early and late promoters were previously described (Yang, Z., Reynolds, S. E., Martens, C. A., Bruno, D. P., Porcella, S. F., and Moss, B., 2011. J. Virol. 85, 9899-9908). Intermediate and intermediate/late promoter logos were derived from 10 intermediate promoters with negligible late activities and 10 promoters with the highest late activities, respectively.

Introducing the author

Moss_Zhilong1

Zhilong Yang
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA

About the research

Cascade regulation of vaccinia virus gene expression is modulated by multistage promoters

Zhilong Yang, Liliana Maruri-Avidal, Jerry Sisler, Carey A. Stuart and Bernard Moss

Virology, Volume 447, Issues 1-2, December 2013, Pages 213-220

 

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