The human herpesvirus-8 (Kaposi’s sarcoma-associated herpesvirus) ORF 40/41 region encodes two distinct transcripts

David P. AuCoin1 and Gregory S. Pari1

Department of Microbiology and the Cell and Molecular Biology Program, School of Medicine, University of Nevada–Reno, Howard Building, Reno, NV 89557, USA1

Author for correspondence: Gregory Pari. Fax +1 775 784 1620. e-mail gpari{at}med.unr.edu


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The human herpesvirus-8 (HHV-8) locus encoding ORFs 40/41 is a candidate homologue for the Epstein–Barr virus BBLF 2/3 gene, which encodes the putative primase-associated factor. Northern blot data revealed that two transcripts originated from the HHV-8 ORF 40/41 region. The sizes of these transcripts (2.2 and 0.7 kb) suggested that one transcript was the result of a spliced form of ORFs 40 and 41 and the second transcript originated from a region within ORF 41. cDNA sequence and 5' RACE analysis revealed the removal of an intron between ORFs 40 and 41 and a transcriptional start site 82 nt upstream of ORF 40. The start of transcription for the smaller transcript was mapped to within ORF 41. Regions upstream of the transcriptional start sites were subcloned into a luciferase reporter vector, and transient luciferase assays indicated that distinct promoters drive the expression of each transcript.


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Human herpesvirus-8 (HHV-8), a member of the genus Rhadinovirus of the subfamily Gammaherpesvirinae, is the probable cause of Kaposi’s sarcoma (KS) (Chang & Moore, 1996 ). In a recent study of patients with clinical signs of KS, more than 95% were infected with HHV-8 (Ambroziak et al., 1995 ; Gao et al., 1996 ; Huang et al., 1997 ; Kedes et al., 1997 ; Moore & Chang, 1995 ; Schalling et al., 1995 ; Whitby et al., 1998 ). HHV-8 has been linked to primary effusion lymphoma, an AIDS-related malignancy, and also to a lymphoproliferative disorder, multicentric Castleman’s disease (Soulier et al., 1995 ).

Herpesvirus lytic DNA replication requires virus-encoded transacting factors, referred to as the ‘core’ replication machinery, which have been identified in several herpesviruses by using a co-transfection replication assay first developed for herpes simplex virus type 1 (HSV-1) (Challberg, 1986 ; Wu et al., 1988 ). Homologues of the core proteins have been identified in all herpesviruses studied to date.

Comparison of herpesvirus homologues of primase-associated factor (PAF) genes indicated that the human cytomegalovirus (HCMV) UL102 and HSV-1 UL8 loci encode unspliced PAF transcripts (Smith & Pari, 1995 ; Wu et al., 1988 ). In contrast, the corresponding loci in Epstein–Barr virus (EBV) and HHV-8 contain two short ORFs. In EBV, these ORFs are spliced to form a continuous ORF (Fixman et al., 1995 ). It is presumed that the transcript encoding the HHV-8 ORFs is spliced similarly. The HHV-8 ORFs 40 and 41 are respectively 1373 and 617 nt in length. A recent report described the identification of a spliced transcript for ORFs 40/41 (Wu et al., 2001 ); however, a detailed characterization of the ORF 40/41 region has not been undertaken.

In an effort to characterize the transcript encoding ORFs 40/41, we generated two riboprobes. RP-A is entirely within ORF 40 and RP-B is entirely within ORF 41 (Fig. 1a, bottom). Northern blots revealed that a single 2·2 kb transcript was detected when the RP-A riboprobe was used to probe RNA from BC3 cells that were induced with tetradecanoylphorbol acetate (TPA)/sodium butyrate (Fig. 1a, lane 2).



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Fig. 1. Transcript analysis of the ORF 40/41 region and identification of an intron between ORFs 40 and 41. (a) Total cellular RNA was isolated from TPA/sodium butyrate-treated BC3 cells and probed with a riboprobe complementary to ORF 40 (RP-A; lanes 1 and 2) or ORF 41 (RP-B; lanes 3 and 4). Lanes: 1 and 3, RNA from untreated BC3 cells; 2 and 4, RNA from TPA/sodium butyrate-treated BC3 cells harvested 8 h post-treatment. The arrowhead shows the presence of a 0·7 kb transcript when blots were hybridized with RP-B. (b) Total cellular RNA from BC3 cells treated for 8 or 16 h with TPA/sodium butyrate was used in an RT–PCR with primers that flanked the putative stop codon for ORF 40 and the putative translation start of ORF 41. The forward primer was an oligonucleotide that was 247 bp upstream of the putative translation stop of ORF 40, at nt 61678. The reverse primer was 35 bp downstream of the putative start of translation for ORF 41, at nt 61827. PCR products were separated on an agarose gel and visualized using a transilluminator. Lanes: 1, PCR of genomic HHV-8 DNA; 2 and 3, RT–PCR of BC3 RNA treated with TPA/sodium butyrate for 8 (2) or 16 (3) h; MW, molecular mass markers. The arrowhead indicates the faster migrating band, where 126 bases have been spliced out of the mRNA. The sequence of the ends of the intron region for ORF 40/41 is shown at the bottom. The underlined sequence shows the 5' and 3' ends of the 126 bp intron; the bold sequence shows the 3' and 5'ends of the spliced exon region.

 
Northern blot data obtained using the probe RP-A indicated that the putative PAF transcript was a 2·2 kb message and that this was the only mRNA transcribed from this region. A second riboprobe was generated complementary to a region further downstream of ORF 40, within the putative ORF 41 coding region. This was done in order to confirm that the PAF transcript included ORF 41, or at least mapped to this region, and to determine whether the ORF 41 region could be the origin of a second, independent transcription unit. When the probe RP-B was used, Northern blots revealed that, in addition to the 2·2 kb RNA species detected with the RP-A probe, a second, smaller, less abundant transcript, approximately 0·7 kb in length, was detected (Fig. 1a, lane 4). Both transcripts were considerably more abundant in RNA samples isolated from TPA/sodium butyrate-treated BC3 cells. These data suggested that a second transcript originated within the ORF 40/41 region and that the start of transcription for the 0·7 kb RNA species was downstream of the ORF 40 ATG.

In order to investigate the possibility that an intron is present between ORFs 40 and 41, we performed RT–PCR on RNA isolated from BC3 cells after 8 and 16 h treatment with TPA/sodium butyrate. The results of the RT–PCR indicated that the amplified cDNA product was approximately 300 bp in length, whereas the PCR product resulting from amplification of genomic DNA was the predicted size of 458 bp (Fig. 1b; compare lane 1 to lanes 2 and 3). Sequence analysis revealed that the 2·2 kb putative PAF mRNA is a spliced transcript and that a 126 bp intron (nt 61658–61784) is present within the genomic sequence (Fig. 1b, bottom). This splicing event results in a 2 kb ORF, where the ORF 40 stop codon is removed and the 5' portion of ORF 40 is spliced, in the same reading frame, to the ORF 41 ATG. This new ORF has the capacity to encode a 75 kDa protein. The identification of an intron in the ORF 40/41 region confirms the earlier report of Wu et al. (2001) .

A time-course experiment was performed to determine the relative abundance of the two transcripts. RNA from BC3 cells was harvested and hybridized with riboprobe RP-B so that accumulation of the 2·2 and 0·7 kb transcripts could be evaluated at various times after treatment with TPA/sodium butyrate. Both transcripts were detected as early as 4 h after TPA/sodium butyrate treatment (Fig. 2a, lane 2). The 2·2 kb transcript was most abundant at 8 and 16 h after TPA/sodium butyrate treatment (Fig. 2a, lanes 3 and 4); however, the 0·7 kb transcript was not detected at 24 h after TPA/sodium butyrate treatment (Fig. 2a, lane 5). The putative PAF transcript was detected in the presence of phosphonoformic acid (PFA), indicating that ORF 40/41 encodes a typical early gene product (Fig. 2a, lane 6). When RNA from TPA/sodium butyrate-treated cells incubated with PFA was harvested at 8 and 16 h, the 0·7 kb transcript was also present, indicating that it is also an early gene (Fig. 2a, lanes 7 and 8). A faint band was observed in the RNA sample harvested from cells in the absence of TPA/sodium butyrate treatment, indicating a low level of lytic replication within the cell population (Fig. 2a, lane 1).



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Fig. 2. Kinetics of expression of the 2·2 and 0·7 kb transcripts. (a) BC3 cells were treated with TPA/sodium butyrate and total cellular RNA was harvested at various times post-treatment and hybridized with RP-B riboprobe. Lanes: 1, no treatment; 2–5, cells treated for 4 (2), 8 (3), 16 (4) or 24 (5) h; 6–8, cells subjected to a 1 h pre-incubation with PFA followed by continual incubation with PFA throughout TPA/sodium butyrate treatment for 24 (6), 8 (7) or 16 (8) h. Arrowheads indicate the 2·2 and 0·7 kb RNA species. (b) Total cellular RNA was harvested from TPA/sodium butyrate-treated BC3 cells incubated in the presence of increasing amounts of cycloheximide and hybridized with RP-B. RNA was harvested from: TPA/sodium butyrate-treated cells harvested 8 h post-treatment (lane 1); untreated BC3 cells (2); TPA/sodium butyrate-treated cells, incubated with 10 (3), 20 (4) or 500 (5) µg/ml cycloheximide and harvested 8 h post-treatment. Blots were re-hybridized with a probe for 18S rRNA to show that equal amounts of RNA were loaded on the gel (below).

 
We also examined the accumulation of PAF mRNA in the presence of cycloheximide. RNA from BC3 cells treated with TPA/sodium butyrate and incubated with increasing concentrations of cycloheximide was hybridized with the riboprobe RP-B. A concentration of 20 µg/ml cycloheximide inhibited the expression of transcripts from the ORF 40/41 locus (Fig. 2b, lane 4). Cycloheximide treatment also eliminated the expression of the 0·7 kb transcript in RNA samples harvested at 8 h after TPA/sodium butyrate treatment, when the 0·7 kb transcript is most abundant in the absence of cycloheximide (Fig. 2b, compare lanes 1 and 4).

In order to locate the start of transcription for ORF 40, we performed 5' RACE analysis on RNA isolated from BC3 cells after 8 h of TPA/sodium butyrate treatment. Amplified cDNA ends were ligated into pGEM-Teasy and 15 ampicillin-resistant colonies were selected and sequenced. Twelve of the 15 resulting plasmids were identical in sequence, indicating that the start of transcription was 82 nt upstream of the putative ATG for ORF 40, mapping to nt 60226 in the HHV-8 genome (Fig. 3a).



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Fig. 3. Promoter activity associated with regions upstream of ORF 40/41 and ORF 41a. (a) Schematic transcription map of the ORF 40/41 region. The genomic locations of ORFs 40 and 41 and the positions of the two transcripts identified by Northern blot analysis are shown. Coordinates for the start of transcription of the 2·2 and 0·7 kb mRNA species are given. The location of the 126 bp intron between ORFs 40 and 41 is also shown. (b) Schematic representation of the ORF 40/41 region, showing the DNA fragments subcloned into luciferase reporter vector pGL2-Basic to make the plasmid constructs p40/41-Luc, p41a-Luc and pCtl-Luc. The transcription initiation sites for 40/41 and 41a mRNA (downstream of the putative ORF 41 ATG) are also shown. (c) Identification of promoter regions for 40/41 and 41a. Vero cells were transfected with luciferase reporter constructs, cells were harvested at 48 h post-transfection and luciferase activity was then measured. Error bars are standard deviations from three experiments. All values were normalized against a Renilla luciferase internal control vector activity, which was included in all transfections. RLU, Relative light units.

 
5' RACE analysis was also performed with primers downstream of the putative ATG for ORF 41. Sequence analysis of 10 plasmids revealed that the start of transcription mapped to nt 61871, 40 nt downstream of the ATG of ORF 41 (which actually encodes a methionine as part of ORF 40). Inspection of the genomic sequence identified a potential ATG at nt 61908, 36 nt downstream of the start of transcription for the 0·7 kb transcript (Fig. 3a). We have designated this smaller transcript the ORF 41a transcript.

Since the start of transcription for the ORF 41a transcript was located within the spliced ORF 40/41 transcript, we investigated the possibility that the upstream region of ORF 41a could act as a promoter that directed transcription of the ORF 41a transcript. We subcloned a 439 bp DNA fragment, corresponding to nt 61372–61811, into the luciferase reporter vector pGL2-Basic to make p41a-Luc (Fig. 3b). In addition to this construct, two others were made: p40/41-Luc contains the upstream region (803 bp) for ORF 40/41, and pCtl-Luc contains a DNA fragment from within the ORF 41 coding region (Fig. 3b). These constructs were transfected into Vero cells and luciferase activity was measured. Vero cells were used because transfection efficiencies for these cells are considerably higher than those achieved in BC3 cells (Duan et al., 2001 ). The construct containing the DNA fragment subcloned from the coding region of ORF 41, pCtl-Luc, failed to show any significant luciferase activity (Fig. 3c). However, the two constructs that contained DNA fragments upstream of either ORF 40/41 (p40/41-Luc) or ORF 41a (p41a-Luc) showed strong promoter activity in Vero cells, as demonstrated by a significant increase in luciferase activity over the control construct (Fig. 3c). These data demonstrate that the region upstream of the ORF 41a transcription initiation site has significant promoter activity and suggest strongly that this region directs expression of the ORF 41a transcript.

Although the loci encoding herpesvirus replication genes have been known in many different systems for some time, the regulation and pattern of expression of many of the core replication proteins have not been investigated. The gene encoding the PAF subunit of the helicase–primase complex appears to be the most divergent among the well-characterized herpesviruses. The PAF subunit is an essential protein for herpesvirus origin-dependent DNA replication and is one of three proteins that comprise the helicase–primase complex (Fixman et al., 1992 ; Pari & Anders, 1993 ; Wu et al., 1988 ). In HSV-1, PAF (UL8) has been shown to form a complex with the helicase–primase subunits, which is presumably situated at the head of the replication fork. In vitro assays have shown that the UL8 protein is indispensable for helicase activity (Tenney et al., 1994 ). Previous studies have demonstrated that the UL8 protein functions to increase efficiency of primer synthesis by UL5/UL52 (Barnard et al., 1997 ; Dodson & Lehman, 1991 ; Tenney et al., 1994 ). As a complex, these three proteins are capable of helicase, DNA-dependent ATPase/GTPase and primase activities (Crute & Lehman, 1991 ; Crute et al., 1989 ; Parry et al., 1993 ). For HCMV, the PAF gene (UL102) was thought originally either to produce a spliced transcript or to have a complex pattern of expression (Pari & Anders, 1993 ). Characterization of the mRNA and a detailed analysis of the transcription unit revealed that the UL102 transcript is a 2·7 kb unspliced message detected in the presence of PFA as early as 24 h post-infection (Smith & Pari, 1995 ).

In EBV, the PAF transcript originates from the BBLF 2/3 locus (Fixman et al., 1992 ). Although the EBV PAF transcript itself has not been identified, RT–PCR analysis has confirmed that it is expressed as a spliced transcript (Fixman et al., 1995 ). For HHV-8, the arrangement of ORFs for the putative PAF locus resembles that of EBV more closely than that of HCMV. The position of BBLF2 and BBLF3 in EBV is similar to the arrangement of ORFs 40 and 41 in the HHV-8 genome. It is clear from the Northern blot results and RT–PCR analysis that the putative PAF transcript is a 2·2 kb spliced transcript.

We have identified putative promoter regions for the spliced ORF 40/41 and ORF 41a transcription units. Transient transfection assays indicated that the promoter for ORF 41a lies within the ORF 40/41 coding region. The putative 41a promoter, as well as the spliced 40/41 promoter, showed a significant increase in basal promoter activity over a control construct. The strong basal activity of the ORF 40/41 and ORF 41a promoter constructs in Vero cells was somewhat surprising. It may be due to the unregulated control of these promoters in cells not permissive for HHV-8. Nevertheless, in Vero cells, the control construct failed to show any significant luciferase activity, whereas regions upstream of ORFs 40 and 41a were highly active, indicating that these upstream regions can act as strong promoters.

At this time, we do not know whether the ORF 41a mRNA encodes a functional protein. The 41a transcript is regulated in a similar manner to the spliced 40/41 transcription unit. This suggests that 41a may encode a protein involved in DNA replication and that regulation of DNA replication in HHV-8 may be unlike that in other gammaherpesviruses. Studies are under way to determine the role, if any, of ORF 41a in the growth of HHV-8.


   Acknowledgments
 
This work was supported by the NIH grant CA85164.


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Received 13 August 2001; accepted 18 September 2001.