Genome-Wide Analysis of the 5′ and 3′ Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts

Abstract

Poxviruses are large DNA viruses that encode a multisubunit RNA polymerase, stage-specific transcription factors, and enzymes that cap and polyadenylate mRNAs within the cytoplasm of infected animal cells. Genome-wide microarray and RNA-seq technologies have been used to profile the transcriptome of vaccinia virus (VACV), the prototype member of the family. Here, we adapted tag-based methods in conjunction with SOLiD and Illumina deep sequencing platforms to determine the precise 5 and 3 ends of VACV early mRNAs and map the putative transcription start sites (TSSs) and polyadenylation sites (PASs). Individual and clustered TSSs were found preceding 104 annotated open reading frames (ORFs), excluding pseudogenes. In the majority of cases, a 15-nucleotide consensus core motif was present upstream of the ORF. This motif, however, was also present at numerous other locations, indicating that it was insufficient for transcription initiation. Further analysis revealed a 10-nucleotide AT-rich spacer following functional core motifs that may facilitate DNA unwinding. Additional putative TSSs occurred in anomalous locations that may expand the functional repertoire of the VACV genome. However, many of the anomalous TSSs lacked an upstream core motif, raising the possibility that they arose by a processing mechanism as has been proposed for eukaryotic systems. Discrete and clustered PASs occurred about 40 nucleotides after an UUUUUNU termination signal. However, a large number of PASs were not preceded by this motif, suggesting alternative polyadenylation mechanisms. Pyrimidine-rich coding strand sequences were found immediately upstream of both types of PASs, signifying an additional feature of VACV 3-end formation and polyadenylation.High-throughput cDNA sequencing has enabled the genome-wide profiling of the transcriptomes of eukaryotic (58) and microbial organisms (57) and of complex DNA viruses (37,61). We recently applied RNA-seq technology for whole-transcriptome analysis of vaccinia virus (VACV) (61), a poxvirus that replicates and transcribes its 195-kbp DNA genome within the cytoplasm of infected cells (42). Early transcripts, synthesized before viral DNA replication, were mapped to 118 closely spaced open reading frames (ORFs), and additional transcripts, synthesized only after DNA replication, were mapped to 93 ORFs. Whole-transcriptome analysis, however, may not delineate the ends of RNAs to high precision or delineate overlapping transcripts. Here, we adapted tag-based RNA-seq methods (27,39,56) to map the 5Ј and 3Ј ends of early VACV transcripts and determine putative regulatory sequences for transcription start sites (TSSs) and polyadenylation sites (PASs).VACV and other poxviruses package a complete virus-encoded transcription system, which allows the early class of mRNAs to be synthesized immediately after entry into the cytoplasm (42). De novo synthesis of proteins and DNA are required to transcribe additional genes, which are subdivided into intermediate and late postreplication (PR) classes. The three...

Expression Profiling of the Intermediate and Late Stages of Poxvirus Replication

“…Intermediate and late RNAs were transcribed from both DNA strands but occurred in clusters on the same strand. Overall, the results suggested similar numbers of intermediate and late mRNAs, as predicted from studies of the defect in protein synthesis resulting from repression of a late transcription factor (24). However, due to the close spacing of ORFs, the extensive read-through of transcripts, and the possibility that many ORFs have hybrid promoters with early, intermediate, or late elements, it was difficult to classify individual ORFs with high confidence from the RNA-seq data alone.…”

“…All cells were cultured at 37°C in a 5% CO 2 atmosphere. Preparation, purification, and titration of VACV were described previously (11,24). Where indicated in the text and figures, 100 g/ml cycloheximide (CHX) or 40 g/ml cytosine arabinoside (AraC) was added to the medium.…”

“…HeLa S3 cells were maintained in minimum essential medium with spinner modification (Quality Biological, Gaithersburg, MD) supplemented with 5% equine serum (HyClone, Logan, UT). Infection of HeLa S3 cells was as described previously (24). BS-C-1 cells were cultured in minimum essential medium with Earle's balanced salts (Quality Biological) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 g/ml penicillin-streptomycin.…”

“…The Western Reserve (WR) strain of VACV (American Type Culture Collection VR-1354) and recombinant forms were used. The recombinant virus vRO-G8R with isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible late transcriptional factor G8R was described previously (24,26). HeLa S3 cells were maintained in minimum essential medium with spinner modification (Quality Biological, Gaithersburg, MD) supplemented with 5% equine serum (HyClone, Logan, UT).…”

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 .…”