Simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA sequencing

Abstract

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 approximately 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-kappaB cascade, apoptosis, signal transduction, and ligand-mediated signaling, which likely represent the host response to invasion.

Fig. 1.

VACV genome-wide transcriptome maps during the first 4 h of infection. The number of sequence reads per nucleotide was determined from the WTA-B time course and displayed over the ORF map of the entire VACV genome. The counts above the line map to the upper (rightward) DNA strand and counts below the line map to the lower (leftward) DNA strand. The highest read counts are off-scale in the 1- to 4-h samples for display purposes. The HindIII restriction map of the VACV genome is shown at the bottom for reference purposes.

Fig. 2.

VACV transcriptome of cells infected in the presence of AraC or CHX. HeLa cells were infected for 2 h in the presence of AraC or CHX, and genome-wide transcriptome maps are displayed as in Fig. 1.

Fig. 3.

HindIII D region of VACV transcriptome. (A) Enlargement of the HindIII D region of the AraC and 4-h transcriptome maps in Figs. 1 and 2. VACVWR ORFs 106–118 correspond to D1–D13 using the Copenhagen nomenclature. The ORFs in green indicate early genes and the ORFs in red indicate late genes. (B) Single-base-resolution plot of the start region of ORF 106 (D1R). Asterisks indicate the transcriptional start sites of D1R previously identified by primer extension.

Fig. 4.

Cluster analysis of the temporal expression of VACV mRNAs. (A) Heat-map representation of the normalized read counts of VACV ORFs from 0 to 4 h in the absence of drugs. The 13 ORFs that showed extensive read-through from upstream ORFs in the presence of AraC and classified as PR (Table S3) were excluded from the analysis. Colors from blue to yellow to red indicate increased numbers of the normalized read counts of each ORF. The clusters labeled C1.1 and C1.2 contain genes expressed early, whereas C2 contains genes expressed after DNA replication. (B) Line plots of read counts corresponding to A. (C) Map of VACV genome showing ORFs transcribed to the right (above line) and to the left (below line) colored red, green, or black to represent cluster 1.1 (E1.1), cluster 1.2 (E1.2), and cluster 2 (PR). (D) Assigned functions of genes in the different expression classes.

Fig. 5.

Analysis of cellular mRNA changes during VACV infection. (A) Heat maps. The read counts of each cellular mRNA were normalized by the sum of the total reads, and the fold changes from 0 time were calculated. The heat maps of WTA-A and WTA-B were generated according to the order of the fold changes at 4 h postinfection. Colors from yellow to blue indicate down-regulated cellular genes; colors from yellow to red indicate up-regulated cellular genes. (B) Functional classification of genes with RNAs that increased at least 2-fold in both WTA-A and WTA-B. The up-regulated genes were analyzed in DAVID 6.7β (SI Materials and Methods), and the PANTHER biological process terms with a P value < 0.05 were chosen and displayed. The percentages of the genes of total up-regulated genes in overrepresented categories are shown at 2 h. Up-regulated genes are: NF-κB cascade: CARD9, LIF, NF-KB2, TNFRSF21, RELB; apoptosis: RELT, CARD9, CXCR4, EMP1, LIF, NFKB2, SPHK1, TNFRSF10D, TNFRSF21, RELB; inhibition of apoptosis: RELT, LIF, NFKB2, SPHK1, RELB; signal transduction: EPHA8, GPR3, GPR87, RELT, S100A13, STARD13, ARMC5, CAPN2, CLCF1, CARD9, CXCR4, DGKD, DOK3, EGFR, GJB3, GNRH1, HBEGF, IL1RAP, IRAK2, JUN, KALRN, LIF, NFKB2, PLG2G16, PTPRH, SLC1A5, SPHK1, TNFRSF10D, TNFRSF21, UCN2, RELB; and ligand-mediated signaling: GPR3, CLCF1, GNRH1, HBEGF, SLC1A5, UCN2, LIF.