Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes

Abstract

Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. Almost all of the genes were transcribed, and for 62 of these, this is the first empirical evidence of expression. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate‐early expression. Although a similar kinetic class has been described for other virus families, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other 3 kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome‐wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression. This work was supported by National Institutes of Health grants ROI‐AI‐56268 and HHSN266200400124C.

Modification of promoter spacer length in vaccinia virus as a strategy to control the antigen expression

“…Two categories of VACV early genes have been defined based on their temporal expression (Assarsson et al, 2008;Yang et al, 2010); their promoter motif (consensus sequence) and their promoter spacers were also recently reported (Yang et al, 2011a). To define the role of the promoter spacer in the optimization of early antigen expression, we designed two new promoters, LEO99 and LEO160, with distinct spacer lengths and compared them with the previously reported LEO (termed here LEO38) promoter .…”

Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling

“…Nine proteins from which HLA ligands were derived are among the 29 previously described immunogenic early proteins (10), and 4 proteins, B19R, E5R, G5.5R, and B15R, were newly identified in this study to contain relevant human CTL epitopes (Table VI). The proteins bearing HLA ligands functionally belong to two groups: proteins with immunomodulatory or host range and virulence function (B8R, B15R, B19R, C7L, and F12L (51,56,57)), and proteins functionally connected to DNA replication or transcription (E5R, A48R, G5.5R, A23R, D12L, and J6R (51)). The function of O1L is unknown.…”

“…This was surprising, because it had been shown for cells other than DC that proteins with late temporal expression can already be detected 4 h after infection, 6.5 h earlier than the time point at which the infected B-LCL were harvested for HLA ligand purification (43,51). Therefore, we analyzed the levels of viral protein expression and viral HLA ligand presentation in infected B-LCL simultaneously.…”

Simultaneous Quantification of Viral Antigen Expression Kinetics Using Data-Independent (DIA) Mass Spectrometry

“…S3), as has been done previously for transcriptional clustering (14,15). These studies showed that, in general, genes with products that are involved in DNA replication and host interaction are expressed at earlier times, followed by those involved in transcription, with virion components only expressed at later times.…”

“…A separation of early genes into two classes by a cluster analysis of the VACV transcriptome was done first by Assarsson et al by tiling microarray and then Yang et al using deep sequencing of poly(A) RNA (14,15). However, these descriptive data remain unsupported by the current understanding of VACV transcriptional control and more recent studies focus on the three known classes of promoter (early, intermediate and late) and the consequences of these elements being combined (16,37).…”