The lytic transcriptome of Kaposi's sarcoma-associated herpesvirus reveals extensive transcription of noncoding regions, including regions antisense to important genes

Abstract

Genomewide analyses of the mammalian transcriptome have revealed that large tracts of sequence previously annotated as noncoding are frequently transcribed and give rise to stable RNA. Although the transcription of individual genes of the Kaposi's sarcoma-associated herpesvirus (KSHV) has been well studied, little is known of the architecture of the viral transcriptome on a genomewide scale. Here we have employed a genomewide tiling array to examine the lytic transcriptome of the Kaposi's sarcoma-associated herpesvirus, KSHV. Our results reveal that during lytic growth (but not during latency), there is extensive transcription from noncoding regions, including both intergenic regions and, especially, noncoding regions antisense to known open reading frames (ORFs). Several of these transcripts have been characterized in more detail, including (i) a 10-kb RNA antisense to the major latency locus, including many of its microRNAs as well as its ORFs; (ii) a 17-kb RNA antisense to numerous ORFs at the left-hand end of the genome; and (iii) a 0.7-kb RNA antisense to the viral homolog of interleukin-6 (vIL-6). These studies indicate that the lytic herpesviral transcriptome resembles a microcosm of the host transcriptome and provides a useful system for the study of noncoding RNAs.

FIG. 1.

KSHV transcriptome analysis. KSHV tiling microarray data (ordered by genome position) are displayed for 13,746 unique probes. Expression analysis was performed on indicated cells (top line). In each group of microarray data, the color bar describes the fold changes relatively to the appropriate uninfected or infected cells. HUVECs (left panel) and TIME cells (center panel) were infected de novo with BCBL-1-derived KSHV, and RNAs were prepared at the indicated times postinfection. KSHV-infected TIME cells were treated with AdRTA to induce entry into lytic cycle, and RNA harvested at 54 h postinfection (hpi) was similarly examined (center panel, rightmost lanes). (Right panel) RNAs were prepared and analyzed from BJAB and BCBL-1 cells and BCBL-1 cells treated with 600 μM valproic acid (VA) for 48 h.

FIG. 2.

Genomic position of microarray hybridization signal from induced BCBL-1 cells. Approximate genomic positions of microarray hybridization signal are transposed on a KSHV genomic map. Genes in blue are carried by the top strand of the genome and are transcribed left to right. Genes in yellow are carried by the bottom strand and are transcribed right to left. The red bars indicate the genomic location of increased microarray hybridization signal in lytically reactivated BCBL-1 cells.

FIG. 3.

Tiling northern analysis to detect transcripts that are antisense to genes at the left end of the genome. Total RNA from BJAB cells (A) or BCBL-1 cells treated with valproic acid (VA) for 24 h (B) was prepared and resolved by formaldehyde-agarose gel electrophoresis, transferred to nylon membranes, and hybridized to radioactively labeled riboprobes, which detect transcripts that span the regions indicated by the red bars. The Northern data are presented directly below the indicated riboprobe used in each blot (please see supporting information file S1 in the supplemental material for information regarding riboprobe design).

FIG. 4.

Tiling Northern analysis to detect transcripts that are antisense to the latency locus. Total RNA from BJAB cells (A) or BCBL-1 cells (B) treated with valproic acid (VA) for 24 h was subject to Northern blotting analysis using radioactively labeled riboprobes, which detect transcripts that span the regions indicated by the orange bars. The Northern data are presented directly above the indicated riboprobe used in each blot.

FIG. 5.

Northern analysis of ALT RNA in poly(A)-enriched RNA and in multiple cell types. Total or poly(A)-enriched RNA from the indicated cells was subjected to Northern blotting analysis using a radioactively labeled riboprobe that detects ALT RNA. Total RNA or poly(A)-enriched RNA was prepared from BJAB cells, untreated BCBL-1 cells, or BCBL-1 cells treated with valproic acid (VA; 600 μM) for 24 h; untreated BC-3 cells or BC-3 cells treated with valproic acid (600 μM) for 24 h; or SLK cells, untreated iSLK.219 cells, or iSLK.219 cells treated with doxycycline (Dox; 1 μg/ml) for 48 h. Arrows indicate ALT transcript.

FIG. 6.

Fine-structure mapping of ALT RNA. (A) The RNase protection assay (RPA) was performed on total RNA from yeast cells (lane 1), BJAB cells (lane 2), or VA-induced BCBL-1 cells at 24 h postinduction (+RNase; lane 3) using an undigested riboprobe that spans nucleotide positions 120748 to 120283 (−RNase; lane 1). After hybridization of the radiolabeled riboprobe to the indicated RNAs, the probes were subject to a treatment with RNase A or T₁. The protected fragments were analyzed by acrylamide gel electrophoresis. Lane M, molecular size markers. (B) Schematic of ALT RNA transcript structure, based on 5′- and 3′-RACE analyses, RT-PCR analysis, and RPA. The indicated splice site is the same splice site (129219 to 129367) found in the bistronic K14 or vGPCR message. The 3′ end of ALT RNA is coterminal (130546) with the bistronic K14 or vGPCR message. The 5′-RACE clone and the RPA support the conclusion that nt 120297 is the 5′ nucleotide, but other downstream start sites may also exist. Assuming this start site, KSHV pre-miRNAs that are spanned by ALT RNA are indicated by the red symbols, while the pre-miRNAs that are not spanned by the ALT RNA are indicated by the green symbols.

FIG. 7.

Expression of RNA antisense to vIL-6 in lytic infection. RNA extracted from the indicated cell lines was oligo(dT) selected and then analyzed by electrophoresis through a 1% agarose gel, transfer to a solid support, and hybridization with a probe complementary to the antisense region of the vIL-6 gene.