Transcriptional analysis of latent and inducible Kaposi's sarcoma-associated herpesvirus transcripts in the K4 to K7 region

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-2 herpesvirus with a genome containing a long unique coding region (LUR) flanked by GC-rich terminal repeat sequences. The LUR encodes approximately 90 annotated open reading frames (ORFs) with complex patterns of gene expression during viral latency, reactivation, and de novo infection. To identify unannotated KSHV genes, we examined the region between 21,500 and 30,000 bp of the KSHV LUR, representing approximately 8.5 kb of sequence. This region encodes seven known single-exon ORFs (K4, K4.1, K4.2, K5, K6, K7, and PAN), but previous computer analyses have failed to identify additional likely genes in the remaining 5.2 kb. We identified four novel transcripts using Northern blotting, phage library screening, and 5' rapid amplification of cDNA ends analysis in the region between ORFs K4.2 and K7. In vitro analysis of KSHV-infected primary effusion lymphoma cell lines in the presence of 12-O-tetradecanoylphorbol-13-acetate and phosphonoformic acid suggests that one latent transcript is coterminal with the previously annotated K3 gene encoding an ubiquitin-ligase known to downregulate major histocompatibility complex class I expression. This alternatively spliced transcript may contribute to KSHV adaptive immune evasion during latent infection. Other transcripts are inducible, including a 6.1-kb transcript that is the largest transcript found in the KSHV genome to date.

FIG.1.

PCR and Northern blot analyses identify previously unannotated transcripts. (A) Map of KSHV genome nt 21500 to 30000 with letter designations (A to CC) for consecutive 300-bp products. Positive PCR amplifications from a BCP-1 TPA-stimulated cell library (dotted line) and a KS tumor cDNA library (dotted-dashed line) are shown and were used as probes for Northern blot analyses. The remaining probes were generated from PCR products using genomic DNA, and primer pairs that did not amplify with either cDNA library. Northern blot analyses were performed using poly(A) RNAs isolated from BC-1 cells that were untreated or treated with TPA as indicated. Previously annotated viral genes with their orientation along with frnk and vnct direct repeat regions are noted. (B) mRNA from BC-1 cells untreated or treated with TPA, PFA, or a combination of TPA and PFA using Probe H to determine directionality and expression pattern. (C) Expression pattern for T_6.1 and T_1.5 transcripts using Probe L with mRNA from BC-1 cells untreated or treated with TPA, PFA, or a combination of TPA and PFA.

FIG.1.

PCR and Northern blot analyses identify previously unannotated transcripts. (A) Map of KSHV genome nt 21500 to 30000 with letter designations (A to CC) for consecutive 300-bp products. Positive PCR amplifications from a BCP-1 TPA-stimulated cell library (dotted line) and a KS tumor cDNA library (dotted-dashed line) are shown and were used as probes for Northern blot analyses. The remaining probes were generated from PCR products using genomic DNA, and primer pairs that did not amplify with either cDNA library. Northern blot analyses were performed using poly(A) RNAs isolated from BC-1 cells that were untreated or treated with TPA as indicated. Previously annotated viral genes with their orientation along with frnk and vnct direct repeat regions are noted. (B) mRNA from BC-1 cells untreated or treated with TPA, PFA, or a combination of TPA and PFA using Probe H to determine directionality and expression pattern. (C) Expression pattern for T_6.1 and T_1.5 transcripts using Probe L with mRNA from BC-1 cells untreated or treated with TPA, PFA, or a combination of TPA and PFA.

FIG. 2.

Directionality of transcripts determined by riboprobe Northern blotting. (A) mRNA from BC-1 cells untreated or treated with TPA with riboprobes from Probe H to determine directionality of transcripts. (B) BC-1 mRNA from untreated or TPA-treated cells with riboprobes from Probe T to determine directionality of T_6.1 transcript.

FIG. 3.

Lambda phage screening with probes L and M for identification of T_1.5. Drawing depicts nine distinct clones from phage screening with Probe L (gray) and three clones from screening with Probe M (black), all with poly(A) tails at nt 25440.

FIG. 4.

Lambda phage screening with probe X for identification of T_6.1. Schematic aligns the five clones identified from phage screening with Probe X. All clones were coterminal with the PAN transcript, with poly(A) tails at nt 29741. Analysis using the MacVector program (Accelrys, Inc.) found one possible ORF (nt 27887 to 28192) on the plus strand (dashed ORF box) in addition to the already annotated ORFK7 and T_1.5 described in this paper.

FIG. 5.

Lambda phage screening with probe H for identification of T_2.5. (A) Schematic shows alignment to KSHV genome of eight identical clones retrieved from phage screening with Probe H. The previously unidentified transcript encodes all of ORFK3 (nt 18596 to 19617), splices to include 208 bp of ORF70 (nt 20096 to 20304), and then splices to a 70-bp fragment within region H (nt 23770 to 23840). (B to E) Rimessi et al. (24) identified four K3 transcripts, a 1.5-kb immediate-early transcript (B) and three early transcripts of 1.3, 2.5, and 2.4 kb (C to E).

FIG. 6.

In vitro translation of 1.3-kb H phage clone φH-3 expresses MIR1 protein. The excised clone from phage screening was in vitro translated in both directions using either T3 or T7 polymerase along with a plasmid containing ORFK3 (P140) as a positive control. The resultant banding pattern matches between the T3 polymerase-translated H phage clone and ORFK3 control plasmid.

FIG. 7.

Latent ORFK3 transcripts are identified by Northern blotting with H probe. The schematic shows the splicing pattern of the excised H phage clone. mRNA from untreated or TPA-treated BC-1 cells was used for Northern blot analyses along with probes for each portion of the clone: 300 bp of K3, 208 bp of ORF70, or 70 bp of region H. The constitutive T_2.5 transcript encoded by the H phage clone φH-3 is seen by Northern blotting with the H fragment but is obscured by induced transcripts when hybridized with probes from ORFK3 and ORF70 regions or with the entire φH-3 clone. Two additional constitutive bands (T_3.0 and T_4.4) hybridize to the H fragment but were not isolated.