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. 2005 Nov;79(22):14457-64.
doi: 10.1128/JVI.79.22.14457-14464.2005.

Transcripts encoding K12, v-FLIP, v-cyclin, and the microRNA cluster of Kaposi's sarcoma-associated herpesvirus originate from a common promoter

Michael Pearce  1 , Satoko Matsumura, Angus C Wilson
Affiliations

Transcripts encoding K12, v-FLIP, v-cyclin, and the microRNA cluster of Kaposi's sarcoma-associated herpesvirus originate from a common promoter

Michael Pearce et al. J Virol. 2005 Nov.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three malignancies associated with AIDS and immunosuppression. Tumor cells harbor latent virus and express kaposin (open reading frame [ORF] K12), v-FLIP (ORF 71), v-Cyclin (ORF 72), and latency-associated nuclear antigen (LANA; ORF 73). ORFs 71 to 73 are transcribed as multicistronic RNAs initiating from adjacent constitutive and inducible promoters upstream of ORF 73. Here we characterize a third promoter embedded within the ORF 71-to-73 transcription unit specifying transcripts that encode ORF 71/72 or K12. These transcripts may also be the source of 11 microRNAs arranged as a cluster between K12 and ORF 71. Our studies reveal a complex arrangement of interlaced transcription units, incorporating four important protein-encoding genes required for latency and pathogenesis and the entire KSHV microRNA repertoire.

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Figures

FIG. 1.
FIG. 1.
The latency gene cluster is expressed via a network of overlapping transcripts. (A) Organization of known ORFs, miRNAs, and mRNA transcripts within the major latency cluster of KSHV. Coordinates are based on the prototype BC-1 sequence (44) and correspond to initiation/termination codons (upper set) and exon sequences (lower set). A cluster of 11 miRNA genes, orientated in the direction indicated by an arrow, are located between the body of the K12 ORF (nucleotide 117,970) and the 3′ end of ORF 71 (nucleotide 121,911) (9, 40, 46). RNAP, RNase protection assay probe. A constitutively active promoter (LTc) gives rise to a precursor RNA (dotted line) that undergoes polyadenylation/cleavage and alternative splicing to produce ∼5.7-kb and ∼5.4-kb tricistronic (ORFs 71, 72, and 73) and ∼1.7-kb dicistronic (ORFs 71 and 72) mRNAs (14, 47, 50). Expression of the lytic activator RTA induces a second promoter (LTi) located between ORF 73 and LTc, giving rise to a 5.5-kb mRNA spanning all three ORFs (35). A 2.3- to 2.5-kb spliced mRNA corresponding to ORF K12, encoding multiple isoforms of kaposin, is transcribed during latency from a promoter at the 3′ end of ORF 73 and is induced further during lytic replication (27, 45). Shorter K12 transcripts initiating at 118,758 have also been reported (45). ORF K14 is essentially silent during latency but strongly induced by RTA utilizing promoter elements shared by LTi (11, 25, 30, 35). (B) Primer extension analysis to detect transcripts initiating upstream of ORF 72. Poly(A)+ RNA was isolated from mock (−Dox; lane 2) or Dox-treated (+Dox; lane 3) TRExBCBL1-Rta cells and reverse transcribed in the presence of radiolabeled oligonucleotide primer 2a. Extension products were resolved on an 8% acrylamide-7 M urea denaturing gel and visualized by autoradiography. Lane 1, size marker (in base pairs) prepared from end-labeled HinfI-digested ΦX174 DNA (Promega).
FIG. 2.
FIG. 2.
Characterization of transcripts initiating in the LTd region. (A) Schematic showing the intergenic region between ORFs 72 and 73. The probe used for RNase protection is shown above the ORF map and the primers used for 5′-RLM-RACE are shown below the map. For simplicity, all 5′-RLM-RACE products in panel B are depicted as originating from the common point of origin at nucleotide 123,568. (B) Summary of 5′-RLM-RACE products obtained from uninduced (−Dox) and induced (+Dox) TRExBCBL1-Rta cells using the KSHV-specific primers described for panel A. Twenty-one out of 38 5′-RLM-RACE products sequenced terminated in the highlighted region between nucleotides 123,739 and 123,791. An additional six clones (not shown) contained products corresponding to the splice from 123,776 to 127,813 and terminated at 127,880.
FIG. 3.
FIG. 3.
RNase protection assays to characterize the 5′ and 3′ ends of ORF 71- and 72-associated transcripts. (A) A 373-nucleotide 32P-labeled antisense riboprobe (KSHV nucleotides 123,568 to 123,874 plus vector sequence) was transcribed in vitro using T7 RNA polymerase. Undigested probe is shown in lane 1. Probe was hybridized with 10 μg of total RNA from KSHV-negative human HeLa cells (lane 2), Dox-treated TRExBCBL1-Rta cells (lane 3), mock-treated TRExBCBL1-Rta cells (lane 4), BC-1 cells (lane 5), and BC3 cells (lane 6). After hybridization and RNase digestion, protected fragments were resolved on an 8 M urea-8% polyacrylamide gel and visualized by autoradiography. An open arrow indicates full-length probe. Fragment sizes were calculated from a DNA sequencing ladder run in adjacent lanes (not shown). (B) A 388-nucleotide antisense riboprobe (lane 1) corresponding to KSHV nucleotides 121,820 to 122,205 (plus 2 additional nucleotides), was hybridized to total RNA isolated from HeLa (lane 2) and Dox- or mock-treated TRExBCBL1-Rta cells (lanes 3 and 4).
FIG. 4.
FIG. 4.
LTd promoter can be induced by RTA. (A) RT-PCR analysis. Poly(A)+ mRNA was isolated from TRExBCBL1-Rta cells treated for 16 h with water (−Dox) or doxycycline (+Dox). PCR amplification was carried out using serial dilutions (1-, 10-, 25-, and 50-fold) of the cDNA with primers complementary to KSHV LT and human GAPDH, and the products were resolved on a 2% agarose gel stained with ethidium bromide. Size markers (lane 1) are shown in base pairs. (B) A total of 0.5 μg each of various reporter constructs was electroporated into 1 × 106 HeLa cells, together with 1 μg of an empty expression vector (−) or pCMV-RTA encoding full-length KSHV RTA (+). Luciferase activity was measured 24 h after transfection. Bars represent the mean and standard deviation of three independent transfections. (C) As in panel B, except that reporters were also cotransfected with pCMV-RTAΔAD, encoding a version of RTA truncated at residue 530 to remove the C-terminal activation domain. (D) A total of 1 × 107 TRExBCBL1-Rta cells were transiently transfected with 2 μg of the luciferase reporter construct indicated. After 24 h, the transfected cells were split into two flasks, and either mock or Dox treated. After a further 18 h, cell extracts were prepared and assayed for luciferase activity. (E) Addition of NaB amplifies the RTA response. Transfected TRExBCBL1-Rta cells were induced with Dox (1 μg/ml), NaB (3 mM), or a combination of both drugs for 16 h prior to harvest.
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