Deciphering poxvirus gene expression by RNA sequencing and ribosome profiling

Area of Science:

• Virology
• Molecular Biology
• Genomics

Background:

• Vaccinia virus gene expression analysis is complex due to closely spaced genes and transcriptional read-through.
• Previous studies lacked high-resolution, genome-wide insights into poxvirus gene expression at both transcriptional and translational levels.

Purpose of the Study:

• To perform the first genome-wide, high-resolution analysis of vaccinia virus gene expression at transcriptional and translational levels.
• To map translation initiation sites at single-nucleotide resolution.
• To construct a high-resolution genome-wide map of the vaccinia virus translatome.

Main Methods:

• Genome-wide ribosome profiling to assess mRNA in infected cells.
• Utilizing translation inhibitors to map initiation sites.
• Cluster analysis of ribosome-associated mRNAs.

Main Results:

• Viral mRNA constituted approximately 80% of ribosome-associated mRNA by 4 hours post-infection.
• Ribosome-associated mRNAs were detected for most early, intermediate, and late vaccinia virus genes at specific time points.
• Identified novel putative translation initiation sites, primarily within open reading frames, often associated with short coding sequences.

Conclusions:

• Ribosome profiling offers unprecedented clarity and quantitation for poxvirus gene expression, especially for intermediate and late genes.
• Early gene expression is primarily transcriptionally regulated, while later expression is complicated by transcriptional read-through.
• The study provides a foundational high-resolution map of the vaccinia virus translatome, enabling further research into poxvirus gene regulation.