Deep RNA sequencing was used to simultaneously analyze vaccinia virus (VACV) and HeLa cell transcriptomes at progressive times following infection. VACV, the prototypic member of the poxvirus family, replicates in the cytoplasm and contains a double-stranded DNA genome with ≈200 closely spaced open reading frames (ORFs). The acquisition of a total of nearly 500 million short cDNA sequences allowed construction of temporal strand-specific maps of the entire VACV transcriptome at single-base resolution and analysis of over 14,000 host mRNAs. Before viral DNA replication, transcripts from 118 VACV ORFs were detected; after replication, transcripts from 93 additional ORFs were characterized. The high resolution permitted determination of the precise boundaries of many mRNAs including read-through transcripts and location of mRNA start sites and adjacent promoters. Temporal analysis revealed two clusters of early mRNAs that were synthesized in the presence of inhibitors of protein as well as DNA synthesis, indicating that they do not correspond to separate immediate-and delayed-early classes as defined for other DNA viruses. The proportion of viral RNAs reached 25-55% of the total at 4 h. This rapid change, resulting in a relative decrease of the vast majority of host mRNAs, can contribute to the profound shutdown of host protein synthesis and blunting of antiviral responses. At 2 h, however, a minority of cellular mRNAs was increased. The overrepresented functional categories of the up-regulated RNAs were NF-κB cascade, apoptosis, signal transduction, and ligand-mediated signaling, which likely represent the host response to invasion.human transcriptome | poxvirus transcriptome | RNA-seq | vaccinia virus mRNA | virus-host interaction
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...
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