Department of Microbiology and the Cell and Molecular Biology Program, School of Medicine, University of NevadaReno, 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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Abstract |
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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 EpsteinBarr 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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In order to investigate the possibility that an intron is present between ORFs 40 and 41, we performed RTPCR on RNA isolated from BC3 cells after 8 and 16 h treatment with TPA/sodium butyrate. The results of the RTPCR 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 6165861784) 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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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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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 6137261811, 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 helicaseprimase 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 helicaseprimase 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 helicaseprimase 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, RTPCR 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 RTPCR 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.
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References |
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Received 13 August 2001;
accepted 18 September 2001.