The human herpesvirus-8 ORF 57 gene and its properties

Leonard J. Bello1,2, Andrew J. Davison1, Mark A. Glenn3, Adrian Whitehouse4, Nikki Rethmeier1, Thomas F. Schulz3 and J. Barklie Clements1

Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, UK1
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA2
Department of Medical Microbiology, University of Liverpool, UK3
Molecular Medicine Unit, St James University Hospital, Leeds, UK4

Author for correspondence: J. Barklie Clements. Fax +44 141 337 2236. e-mail b.clements{at}vir.gla.ac.uk


   Abstract
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Human herpesvirus-8 (HHV-8) is a {gamma}2 lymphotropic herpesvirus associated with Kaposi’s sarcoma, a major neoplasm of AIDS patients, and with other AIDS-related neoplasms. The HHV-8 ORF 57 gene is conserved throughout the herpesvirus family and has a herpes simplex virus type 1 homologue, IE63 (also termed ICP27), which is an essential regulatory protein and acts at both transcriptional and post-transcriptional levels. We show that, contrary to the published HHV-8 sequence, which predicts a protein of 275 amino acids, the ORF 57 gene is spliced, contains a single intron and encodes a protein of 455 amino acids. For several gammaherpesviruses examined, the upstream coding exon is 16–17 amino acids in length and is rich in methionine residues. When ORF 57 was fused to the gene for enhanced green fluorescent protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the cellular splicing factor SC-35. Unlike the IE63–EGFP fusion protein, ORF 57–EGFP did not shuttle from the nucleus to the cytoplasm in the presence of actinomycin D. However, ORF 57–EGFP was capable of shuttling from a transfected monkey nucleus to a recipient mouse nucleus in an interspecies heterokaryon assay. These data indicate that HHV-8 ORF 57 and IE63 possess certain common properties.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion
References
 
Human herpesvirus-8 (HHV-8), or Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV; Chang et al., 1994 ), is a focus of intensive research. HHV-8 (reviewed in Schulz, 1998 ) has been classified as a member of the lymphotropic or gammaherpesviruses, and is related to other herpesviruses with oncogenic potential, herpesvirus saimiri (HVS), Epstein–Barr virus (EBV) and the murine herpesvirus MHV-68 (Moore et al., 1996 ; Sunil-Chandra et al., 1992 ). KS is a major neoplasm of AIDS patients. Seroepidemiological studies demonstrate that HHV-8 infection is tightly linked to KS risk (Gao et al., 1996 ; Simpson et al., 1996 ) and that virus infection is associated with two other AIDS-related neoplasms, multicentric Castleman’s disease (Soulier et al., 1995 ) and primary effusion lymphoma (PEL), a rare type of B cell lymphoma also found in HIV-negative individuals (Nador et al., 1995 ). B cell lines that are HHV-8-positive have been established from PEL (Boshoff et al., 1998 ), certain of which are co-infected with EBV (Cesarman et al., 1995 ) and others of which are not (Arvanitakis et al., 1996 ). Viral gene expression in these cells is highly restricted, although up to 5% of the population shows spontaneous reactivation. Following treatment of cells with sodium butyrate or tetradecanoylphorbol acetate (TPA), a cascade of viral gene expression is induced that results in virion production and cell lysis (Renne et al., 1996 ).

Expression of HHV-8 transcripts has been studied in these latently infected B cell lines. Three major classes of viral genes have been distinguished (Sarid et al., 1998 ; Sun et al., 1999 ): class I, from which RNA synthesis is unaffected by TPA and which are considered to represent latent viral genes; class II, which are transcribed at higher levels following TPA treatment; and class III, primarily replication and structural genes, synthesis of which requires TPA induction. In BCLB-1 cells, four candidate genes for the switch from latent to lytic replication genes have been identified that are the first to be expressed after stimulation of cells with TPA (Lukac et al., 1998 ; Sun et al., 1998 ). The product of one of these, gene ORF 50, is able to trans-activate expression of delayed-early viral genes. A second regulatory gene, ORF 57, was also expressed following TPA stimulation, together with the K3 and K5 gene products.

The HVS ORF 57 product has trans-regulatory functions that appear to be mediated at the post-transcriptional level (Whitehouse et al., 1998a ) and is functionally homologous to a key herpesvirus protein that regulates virus–host interactions, the 63 kDa immediate-early (IE) phosphoprotein IE63 (also called ICP27), which has been studied extensively in the prototype alphaherpesvirus, herpes simplex virus type 1 (HSV-1). IE63 (reviewed in Phelan & Clements, 1998 ; Sandri-Goldin, 1998a ) is one of two HSV-1 IE proteins essential for lytic virus replication. The presence of a homologue in every herpesvirus of mammals and birds sequenced so far suggests that aspects of the regulatory role of IE63 are maintained throughout the herpesvirus family. These homologues include EBV BMLF1, which has been shown to act post-transcriptionally, affecting RNA splicing and transport (Buisson et al., 1999 ), UL69 of human cytomegalovirus (HCMV; Winkler et al., 1994 ) and gene 4 of varicella-zoster virus (Defechereux et al., 1997 ). Studies of IE63 have highlighted the multifunctional nature of this protein, which acts at both transcriptional and post-transcriptional levels. Acting post-transcriptionally, IE63 (which has a punctate nuclear distribution; Phelan et al., 1993 ) binds RNA in vivo, enhances RNA 3' processing, inhibits pre-mRNA splicing and may facilitate the nuclear export of HSV-1 transcripts.

Contrary to the published HHV-8 DNA sequence (Russo et al., 1996 ), which predicts an unspliced ORF 57 gene encoding a protein of 275 amino acid residues, the ORF 57 gene is spliced. The ORF 57 gene contains two coding exons and a single intron and is predicted to encode a protein of 455 amino acid residues. When cells were transfected with a plasmid containing ORF 57 fused to a marker enhanced green fluorescent protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with a component of the cellular splicing machinery. In contrast, an N-terminal ORF 57 truncation exhibited a predominantly cytoplasmic distribution. Unlike the HSV-1 IE63–EGFP fusion protein, ORF 57–EGFP did not shuttle from the nucleus to the cytoplasm in the presence of actinomycin D. However, in an interspecies heterokaryon assay, ORF 57–EGFP was capable of shuttling from a transfected monkey nucleus to a mouse cell nucleus.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Cell lines and plasmids.
HBL-6 cells (Russo et al., 1996 ) were maintained at 37 °C in RPMI 1640 supplemented with 20% foetal calf serum, 50 IU/ml penicillin, 50 µg/ml streptomycin and 5% CO2. To induce lytic HHV-8 replication, cells were seeded at 4x105 cells/ml, sodium butyrate was added to 3 mM and cells were harvested 48 h later. For non-induced cells, HBL-6 cells seeded at the same density were cultured without the addition of butyrate and harvested in the same manner. Hep-2 cells were grown in DMEM supplemented with 10% foetal calf serum.

A cosmid DNA library was constructed by using an excised biopsy from a case of classic KS (GK18) and partial digestion with Sau3AI. One clone (cos32) contained ORF 57 and substantial HHV-8 flanking DNA sequences.

Plasmid pEGFP-C1 (Clontech), which utilizes the HCMV IE enhancer–promoter to express a GFP variant, was used for fusion to the PCR products obtained by amplification of cos32 with the U/L primer pair (pKS1) or the U2/L primer pair (pKS3) (see Fig. 1A). Reaction conditions were 28 cycles (30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C) with 1 U of Vent DNA polymerase (New England Biolabs). The PCR products were digested with EcoRI, purified through a MicroSpin S-44HR column and cloned into the EcoRI site of pEGFP-C1. Plasmid IE63-EGFP (a kind gift of Dr P. Lomonte, MRC Virology Unit, Glasgow, UK) contained the entire IE63 coding region cloned into the EcoRI site of pEGFP-C1. Each insert was in frame with the upstream EGFP reading frame.



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Fig. 1. Size comparison of the PCR products formed by using the U/L and U2/L primers and cDNA vs genomic (cosmid) DNA templates. (A) Position of the U, U1, U2 and L primers relative to the published position of ORF 57 at nucleotides 82717–83541 (open box). The positions of the upstream ATG at nucleotide 82069, the stop codon at 82153 and the 3' end of ORF 56 at nucleotide 81964 (open box) are indicated. (B) Analysis of PCR products. PCR products were separated by electrophoresis in 0·8% agarose plus 0·5 µg/ml ethidium bromide. Sizes of marker fragments (M) are indicated. Lanes 1 and 2, U/L primers and cDNA (lane 1) or cos32 DNA (lane 2) template. Lanes 3 and 4, U2/L primers and cDNA (lane 3) or cos32 DNA (lane 4) template.

 
{blacksquare} RNA isolation and DNase treatment.
The RNAzol method was used (Cinna/TEL-TEST) to prepare RNA. Cells were counted, pelleted and lysed by the addition of 0·2 ml RNAzol per 106 cells. Chloroform (0·1 vols) was added and the suspension was mixed by inverting and incubated on ice for 15 min. The tubes were centrifuged for 15 min and an equal volume of 2-propanol was added to the upper, aqueous phase. RNA was pelleted by centrifugation for 15 min and the pellets were washed with 70% ethanol. The RNA was dried and dissolved in water treated with diethyl pyrocarbonate. For cDNA synthesis and PCR experiments, the RNA was first treated with DNase. Accordingly, 5 µg of control or butyrate-induced HBL-6 total RNA was treated with 50 µg RNase-free DNase (Sigma) plus 80 U RNAsin (Promega) in a final volume of 100 µl for 1 h at 37 °C. The RNA was then extracted twice with phenol–chloroform and once with chloroform and precipitated with 0·1 vols 3 M sodium acetate plus 2·5 vols ethanol overnight at -20 °C. The precipitate was pelleted by centrifugation, washed once with 70% ethanol, dried and dissolved in water at a concentration of 0·1 µg/µl.

{blacksquare} First-strand cDNA synthesis, PCR amplification and sequencing.
First-strand cDNA was reverse-transcribed from DNase-treated total RNA with Superscript II reverse transcriptase (Life Technologies) and an ORF 57 gene-specific antisense primer by using the protocol described in the manufacturer's 5' RACE System manual. Second-strand synthesis and the first round of PCR amplification were carried out with a nested 3' gene-specific primer designated L and a 5' primer designated U1 (Fig. 1A). The reaction conditions were 35 cycles (30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C) with 2·5 U Taq polymerase. The PCR product was diluted 100-fold and subjected to a second round of PCR amplification using the L primer again and one of two nested 5' primers designated U2 and U (Fig. 1A). Reaction conditions for the second round of amplification were 25 cycles (30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C) with 2·5 U Taq polymerase. The latter conditions were also used for the single round of PCR amplification of cos32.

The cDNA PCR product with the U2/L primer pair was subjected to two rounds of phenol–chloroform extraction, precipitation with ethanol and digestion with EcoRI. The second EcoRI digest was then extracted with phenol–chloroform, precipitated with ethanol, dissolved in TE, purified with a MicroSpin S-400 HR column (Pharmacia Biotech) and cloned into the EcoRI site of pEGFP-C1.

To locate the ends of the gene ORF 57 mRNA by 5'- and 3'-RACE, poly(A)-selected RNA was purified from induced cells by using an Invitrogen FastTrack 2.0 kit and processed by using a Clontech Marathon cDNA amplification kit. Sequences representing the ends of the mRNA were amplified by using PCR program 1 described in the Marathon protocols. Clontech Advantage cDNA mix was used in conjunction with primers AP1 (supplied with the Marathon kit for 5'- or 3'-RACE), 5' dGACCTGGGTCGAGACAGTGGGGAG 3' (ORF 57 5'-RACE; nucleotides 82460–82437), or primer U (ORF 57 3'-RACE). PCR products were cloned into pGEM-T (Promega) and the ends of the inserts were sequenced.

{blacksquare} Northern blot analysis of RNA.
Total RNA from control or butyrate-treated HBL-6 cells was electrophoresed through a horizontal 1% agarose, 2·2 M formaldehyde gel and blotted onto nylon transfer membrane as described by Brown & Mackey (1997) . After transfer of the RNA to nylon, the blots were UV-irradiated for 2 min and pre-hybridized for 6 h at 42 °C in 5 ml buffer containing 50% formamide, 5x SSC, 25 mM potassium phosphate (pH 7·4), 0·2% SDS, 0·1% Ficoll, 0·1% polyvinylpyrrolidone, 0·1% BSA and 0·2 mg/ml denatured, sonicated salmon-sperm DNA. The pre-hybridization solution was then removed. Two ml fresh solution containing a 32P-labelled hybridization probe (1–2x107 c.p.m./ml) was added and incubation at 42 °C was continued for 20 h. Hybridization probes were generated either by random labelling of the 0·84 kb EcoRI DNA fragment from pKS1, containing the entire published ORF 57 sequence, with [{alpha}-32P]dCTP by using the Random Primer labelling system (Life Technologies) or, in the case of the H3 and NH1 oligonucleotide probes (Fig. 2), by end-labelling with T4 polynucleotide kinase and [{gamma}-32P]ATP. After hybridization, the blots were washed three times in 1x SSC, 0·1% SDS at room temperature for 30 min and twice in 1x SSC, 0·1% SDS at either 65 °C (random labelled probe) or 60 °C (end-labelled probe) for 45 min and exposed to Kodak X-OMAT AR film.



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Fig. 2. Position of the intron found in the DNA region between the U2 and U primers. The HHV-8 sequence from nucleotides 81901 to 82750 is shown with the position and orientation of the U1, U2 and U primers indicated above the sequence and the intron (nucleotides 82118–82225) enclosed within the box. The TAA stop codon for ORF 56 at nucleotide 81965, the ATG at nucleotide 82069 and the ATG start codon for the published version of ORF 57 at nucleotide 82717 are enclosed in boxes. A potential TATA element is indicated by lines above and below the sequence. The position and orientation of the H3 and NH1 hybridization probes used in Fig. 3 are indicated below the sequence.

 


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Fig. 3. Northern blot analysis of RNA from control and HBL-6 cells after induction. The sizes of the transcripts that hybridized with the ORF 57 random-labelled probe or the H3 and NH1 end-labelled probes are indicated. The same blot was used with each of the probes. The order of hybridization was H3 first, NH1 second and ORF 57 last. The blot was stripped and the elimination of labelled bands was confirmed between hybridizations. Exposure time was 5 days with the H3 probe, 7 days with NH1 and 8 h with ORF 57. Lanes 1, 3 and 5 contain 6 µg RNA from non-induced cells. Lanes 2, 4 and 6 contain 6 µg induced-cell RNA.

 
{blacksquare} DNA transfections and shuttling experiments.
Hep-2 cells grown on coverslips were electroporated with 20 µg of appropriate plasmid DNA as described previously (Phelan et al., 1993 ). To examine shuttling in the presence of actinomycin D, the conditions used were similar to those that showed shuttling of IE63 (Phelan & Clements, 1997 ). Actinomycin D (10 µg/ml) was added 24 h after transfection and cells were incubated for a further 3 h and then fixed with formaldehyde (5%, v/v) in PBS containing 2% sucrose, washed three times with PBS and once with water and dried. The coverslips were mounted and examined with a Nikon Microphot-SA microscope adapted with a Digital Pixel CCD digital camera. EGFP fluorescence was examined following excitation at 488 nm. Visualization of SC-35 with an anti-SC35 MAb (Sigma) was performed as described previously (Cooper et al., 1999 ). Captured images were prepared for printing by using Adobe Photoshop.

To examine shuttling in the interspecies heterokaryon assay, 1x106 Cos-7 cells were transiently transfected by using the calcium phosphate precipitation technique with 2 µg pKS3 or pKS1. After 24 h, 2x105 cells were transferred to coverslips in 6-well trays, mixed with 2x105 3T3 cells and left to settle for 2 h in medium containing 50 µg/ml cycloheximide. Cells were washed in PBS and fused by the addition of 2 ml 50% (w/w) polyethylene glycol in PBS for 2 min. After washing, cells were returned to medium containing 50 µg/ml cycloheximide for 1 h, and then fixed with 3% (v/v) formaldehyde in PBS. Staining with 0·5 µg/ml Hoechst dye no. 33258 (Sigma) was for 1 h at 37 °C. Hoechst dye allowed the differentiation between monkey and mouse nuclei. When using this assay to demonstrate shuttling of IE63, monkey cells stain diffusely throughout the nucleus whereas mouse nuclei stain with a distinctive speckled pattern (Mears & Rice, 1998 ).


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
The ORF 57 gene contains a single intron
The published sequence of HHV-8 ORF 57 begins at nucleotide 82717 and terminates at nucleotide 83544, encoding a protein of 275 amino acid residues (Russo et al., 1996 ). Because the HVS ORF 57 counterpart is a spliced gene encoding a protein of 416 amino acids, we considered the possibility that HHV-8 ORF 57 contains an upstream exon. The HHV-8 sequence upstream of the published ORF 57 contains 749 nucleotides unassigned to any of the HHV-8 ORFs identified. Within this unassigned region there are five ATG codons. The first four ATGs, clustered within a region of 33 nucleotides, are in frame with each other and with the downstream ORF 57 but are separated from it by a single stop codon. The ATG cluster begins at nucleotide 82069, 648 nucleotides upstream of the published ORF 57 initiation codon. Thus, removal of the single stop codon by splicing in an appropriate register would generate a new ORF 57 much larger than the published version.

To determine whether a spliced transcript extending from the first of the upstream ATGs to the published ORF 57 is synthesized in HHV-8-infected cells, we utilized RT–PCR to analyse RNA from induced HBL-6 cells. mRNA was reverse-transcribed by using an ORF 57 gene-specific antisense primer (5' dCATGGAATACGGGAGACAC 3', nucleotides 83586–83568) positioned downstream of the ORF 57 termination codon. The first round of PCR amplification was carried out with a nested 3' gene-specific primer (designated L) that overlaps the ORF 57 stop codon (5' dGGTTTGGCAATCCTTAAGA 3', nucleotides 83557–83539) and a 5' primer (designated U1) positioned upstream of the first ATG and 33 nucleotides downstream of a possible TATA box site (see Figs 1A and 2). A second round of amplification was then carried out with the L primer again and one of two nested 5' primers (designated U2 and U). U2 overlaps the first ATG in the unassigned sequence region and U overlaps the first ATG in the published ORF 57 sequence (Figs 1A and 2). The U2, U and L primers each contained a 5' dGTCGAATTC 3' sequence to facilitate incorporation of an EcoRI site used to clone the PCR products.

The cDNA PCR products obtained with the U/L and U2/L primers were compared with DNA PCR products generated by the same primers after amplification of HHV-8 cos32, containing the genomic version of HHV-8 ORF 57 (Fig. 1B). Lanes 1 and 2 contain, respectively, the cDNA and DNA PCR products obtained with the U/L combination of primers. The bands are of similar size, indicating that the published ORF 57, as expected, contains no introns. The cDNA and DNA PCR products obtained with the U2/L combination of primers are shown in lanes 3 and 4, respectively. The U2/L cDNA PCR product is approximately 100 bp smaller than the product obtained with the DNA template, indicating the presence of an intron in the DNA region that lies between the U2 and U primers.

To define the exact boundaries of the intron, the cDNA PCR product obtained with the U2/L primers was cleaved with EcoRI to generate cohesive ends and cloned into the EcoRI site of pEGFP-C1. Three of the cDNA clones obtained were sequenced and the sequences were compared with the published HHV-8 sequence. Analysis of the sequence data confirmed the presence of a single 108 bp intron in the U2/L DNA PCR product with splice donor and acceptor sites at nucleotides 82117 and 82226, respectively (Fig. 2). In the spliced U2/L cDNA, the upstream cluster of four ATGs all remain in frame with the downstream ORF 57 and the single intervening stop codon has been eliminated. These results are consistent with the view that ORF 57 contains an upstream exon and encodes a protein larger than the 275 amino acid residues indicated by the published ORF 57 sequence.

The upstream exon is part of the ORF 57 mRNA
The above results indicate that a spliced transcript that includes both the published ORF 57 and an upstream exon is synthesized in HHV-8-infected cells. However, it is not clear that this transcript represents the true ORF 57 mRNA. ORF 56 lies immediately upstream of ORF 57 and lacks a canonical poly(A) signal, suggesting that the ORF 56 and ORF 57 transcripts are 3'-co-terminal. Thus, the U2/L PCR product sequenced might have represented the 3' end of the ORF 56 mRNA rather than the ORF 57 mRNA. To examine this possibility, the size of the ORF 57 transcript was determined by Northern blot analysis with a labelled probe containing the entire published ORF 57 sequence hybridized with total RNA from uninduced and induced HBL-6 cells (Fig. 3, lanes 1 and 2).

A prominent 1·7 kb ORF 57 transcript, as well as a faint 4·1 kb transcript, were evident in RNA isolated from induced cells (Fig. 3, lane 2). The faint 4·1 kb transcript may represent the 3'-co-terminal ORF 56 transcript discussed above. If so, it is present at a level much lower than that of the 1·7 kb ORF 57 mRNA. A much less abundant 1·7 kb ORF 57 transcript could also be seen in RNA from uninduced cells (Fig. 3, lane 1). The induction of ORF 57 mRNA observed after butyrate treatment is consistent with data from BC-1 cells after TPA induction, which places the ORF 57 gene in transcription class II (Sarid et al., 1998 ). The transcript size detected is, within experimental limits, consistent with the size of 2·0 kb observed by Sarid et al. (1998) with an ORF 57-specific probe. Detection of the 1·7 kb mRNA in uninduced cells probably reflects the spontaneous reactivation of virus gene expression that is observed in a small proportion of cells.

The size of the 1·7 kb ORF 57 transcript is consistent with an mRNA initiated from the region of the HHV-8 genome contained within the U1 primer described in Fig. 2(A). If so, the coding region for ORF 57 begins at the first ATG (nucleotide 82069) in the cluster of four ATGs indicated in Fig. 2(A). To confirm the presence of this ATG in the ORF 57 mRNA, the RNA blot was hybridized with an antisense oligonucleotide (designated H3) that overlaps the ATG at its 3' end and extends 29 nucleotides upstream (see Fig. 2). The same 1·7 kb transcript was observed (Fig. 3, lanes 3 and 4), indicating that the ATG at nucleotide 82069 is included in the ORF 57 mRNA. Hybridization of the RNA blot with an antisense probe (NH1) that overlaps the next upstream ATG (nucleotide 81915) failed to identify any transcripts (Fig. 3, lanes 5 and 6). Taken together, the results lead to the conclusion that the ATG at nucleotide 82069 is the initiation codon for ORF 57 and that the encoded protein contains 455 amino acid residues.

5'-RACE experiments (data not shown) confirmed both the absence of the intron from ORF 57 transcripts and the ATG at nucleotide 82069 as the initiation codon for the ORF 57 gene. The locations of 5' ends in several clones obtained from the major 5'-RACE product differed somewhat from each other, suggesting incomplete reverse transcription or partial mRNA degradation. The 5' end of the sequence extending furthest upstream mapped between nucleotides 82017 and 82024 (CGGCTCAC... in Fig. 2), consistent with use of the upstream TATA box at nucleotides 81974–81980. 3'-RACE experiments (data not shown) showed that ORF 57 transcripts (and, presumably, 3'-co-terminal ORF 56 transcripts) are polyadenylated on the C residue at nucleotide 83636, close downstream from a consensus AATAAA polyadenylation signal at nucleotides 83608–83613.

Table 1 shows amino acid sequence alignments at the proposed N termini for ORF 57 and several other gammaherpesviruses, and DNA sequences containing the different 5' and 3' splicing signals are also shown. For the eight viruses compared, the N-terminal sequence encoded by the upstream exon comprises 16 or 17 amino acids, each containing three or four methionine residues, with the exception of the more distantly related EBV BMLF 1 (from a {gamma}1 herpesvirus), which is 20 amino acid residues in length and contains a single methionine.


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Table 1. Characteristics of splice sites in ORF 57 and counterparts in other gammaherpesviruses

 
The ORF 57 protein, but not an N-terminal truncation, locates to the cell nucleus
Homologues of ORF 57 from other herpesviruses have been shown to encode a nuclear protein implicated in RNA processing and transport. To determine whether the HHV-8 protein is also a nuclear protein, the ORF 57 coding region was cloned downstream of EGFP and the cellular distribution of the fusion protein was ascertained. To this end, the genomic U2/L PCR product obtained by amplification of HHV-8 cos32 was cleaved with EcoRI to generate cohesive ends and inserted into the EcoRI site of pEGFP-C1. Three of the clones obtained were sequenced, and all differed from the published HHV-8 sequence by a T to C change (CAT to CAC) at position 82959 of the published sequence. This change did not alter the amino acid sequence of the ORF 57 protein. One of the clones (designated pKS3) was selected for further study.

Plasmid pKS3 was electroporated into Hep-2 cells and the fluorescence pattern was subsequently evaluated (Fig. 4). The pEGFP-C1 vector was included as a control and, as expected, displayed a fluorescence signal in both the nucleus and cytoplasm (Fig. 4A). The pKS3 plasmid, in contrast, exhibited a fluorescence pattern that was confined to the nucleus (Fig. 4B) and resembled the punctate pattern produced by the HSV-1 IE63 homologue (Phelan et al., 1993 ). The punctate nuclear fluorescence of ORF 57–EGFP co-localized with splicing factor SC-35 (Fig. 4D and E), consistent with similar observations with IE63 (Phelan et al., 1993 ) and HVS ORF 57 (Cooper et al., 1999 ). Due to the high background of punctate SC-35 in Hep-2 cells not expressing ORF 57–EGFP, no evidence was obtained that ORF 57–EGFP expression alone, as is the case for IE63 (Phelan et al., 1993 ) and HVS ORF 57 (Cooper et al., 1999 ), was sufficient to redistribute the cellular splicing components into clumps.



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Fig. 4. Cellular localization of the complete and truncated forms of the ORF 57 protein. The pEGFP-C1 vector (A) and the pKS3 (B) and pKS1 (C) fusion gene constructs were introduced into Hep-2 cells by electroporation and the immunofluorescence patterns of the cells were examined 24 h later. (D) Detection of pKS3–EGFP fusion protein after transfection of Hep-2 cells. (E) Detection of splicing factor SC-35 in cells in the same field. Spots corresponding to co-localization of ORF 57–EGFP and SC-35 are indicated by arrowheads in (D) and (E).

 
Interestingly, when plasmid pKS1, which contained the U/L PCR product (the original ORF 57) cloned downstream of EGFP, was electroporated into Hep-2 cells, the fusion protein was confined mostly to the cytoplasm (Fig. 4C). This result indicates that a nuclear localization signal resides in the N-terminal portion of ORF 57 identified in this study. The small amount of nuclear fluorescence observed with pKS1 was restricted to structures that resemble nucleoli.

ORF 57 can shuttle between the nucleus and cytoplasm
Initially, we used conditions of actinomycin D treatment, determined by Pinol-Roma & Dreyfuss (1992) , where inhibition of transcription interferes with the nuclear import of certain proteins while export continues, and this allows the cytoplasmic accumulation of rapidly shuttling proteins to be visualized. We have previously demonstrated the nucleocytoplasmic shuttling ability of the cellular protein hnRNP A1 and of HSV-1 IE63 in virus-infected cells under these conditions (Phelan & Clements, 1997 ). Indirect immunofluorescence experiments were performed after electroporation of Hep-2 cells with pKS3 or pIE63-EGFP DNAs and, after transfection, cells were treated with actinomycin D and incubated for a further 3 h.

The results in Fig. 5 show that, under these conditions, the ORF 57–EGFP fusion protein does not shuttle from the nucleus to the cytoplasm (compare panels C and D), in contrast to the IE63–EGFP fusion protein (compare panels A and B). The nucleocytoplasmic shuttling of IE63–EGFP is consistent with the observation that IE63 itself is capable of shuttling under conditions of actinomycin D treatment when expressed from a transfected plasmid (Sandri-Goldin, 1998b ).



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Fig. 5. HHV-8 ORF 57–EGFP does not shuttle between the nucleus and cytoplasm after treatment with actinomycin D. Hep-2 cells were electroporated with the appropriate EGFP plasmid DNA. After 24 h, cells were treated with actinomycin D for 3 h, fixed and examined for expression of EGFP. (A)–(B) Cells transfected with IE63–EGFP in the absence (A) or presence (B) of actinomycin D. (C)–(D) Cells transfected with pKS3–EGFP in the absence (C) or presence (D) of actinomycin D.

 
In a further test of the shuttling ability of ORF 57–EGFP, we used a sensitive interspecies heterokaryon assay that Mears & Rice (1998) used to identify C- and N-terminal regions involved in shuttling of IE63. Cos-7 monkey cells transfected with pKS3 were fused with untransfected 3T3 cells (mouse) in the presence of cycloheximide. The appearance of ORF 57–EGFP in mouse nuclei of interspecies heterokaryons would indicate that the protein has shuttled out of the transfected monkey nuclei into untransfected mouse nuclei. To differentiate monkey nuclei from mouse nuclei, cells were stained with Hoechst 33258; monkey nuclei stain relatively diffusely with this reagent whereas mouse nuclei exhibit a characteristic speckled pattern. In Fig. 6, a heterokaryon with a distinctively speckled recipient-cell mouse nucleus is indicated. Fig. 6(B) shows fluorescence of pKS3-EGFP in the same heterokaryon, and fluorescence is seen in both donor and recipient nuclei. Examination of numerous interspecies heterokaryons showed that nearly all of those that contained ORF 57–EGFP in their monkey nucleus also displayed ORF 57–EGFP in the mouse nucleus. Thus, the fusion protein was capable of shuttling from the donor transfected nucleus to the recipient nucleus. The ability to detect ORF 57–EGFP shuttling in the heterokaryon assay but not after actinomycin D treatment of transfected cells is observed with many shuttling proteins, including all the nucleocytoplasmic transport receptors such as cyclin B1 (Hagting et al., 1998 ). To demonstrate shuttling following actinomycin D treatment, protein re-import into the nucleus has to be transcription-dependent.



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Fig. 6. HHV-8 ORF 57–EGFP shuttles between the nucleus and cytoplasm in an interspecies heterokaryon assay. Cos-7 monkey cells were transiently transfected with 2 µg pKS3 DNA. After 18 h, 3T3 mouse cells were plated onto the cells in the presence of cycloheximide. The cells were fused by the addition of polyethylene glycol, fixed, stained with Hoechst 33258 dye and examined for EGFP fluorescence. The Hoechst dye stains monkey cells diffusely throughout the nuclei while mouse nuclei stain with a distinctive speckled pattern. (A) Hoechst 33258-stained nuclei. (B) ORF 57–EGFP fluorescence in cells in the same field.

 
These data indicate that the HHV-8 ORF 57 gene product and its distantly related HSV-1 IE63 homologue possess common properties. The amino acid conservation between IE63 and its homologues in other herpesviruses is primarily a feature of the C-terminal portion, a zinc finger-like region in IE63 (Vaughan et al., 1992 ), in which six amino acids are positionally conserved, several of which are essential for IE63 function (Brown et al., 1995 ). Comparisons among members of the herpesvirus family have allowed an understanding of the origins of herpesvirus genes, identified core gene products and facilitated the functional analysis of virus gene products. Studies of molecular evolutionary history indicate that branches giving rise to the alpha- and gammaherpesviruses diverged around 200 million years ago (McGeoch et al., 1995 ). Comparisons between herpesvirus IE63 homologues are therefore likely to facilitate important insights into protein function in the context of the different virus–host relationships, indicating whether they possess similar, additional or different functions.


   Acknowledgments
 
We are grateful to Tenovus-Scotland for an award to support L.J.B. and for funding from the Medical Research Council (G9623413). Part of the sequencing provision was provided by an award from the Wellcome Trust (046745/2/96/ZMP/NOS/JS). We thank Charles Cunningham and Moira Watson for technical support. A.J.D. is a member of the Medical Research Council Virology Unit.


   References
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Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 15 July 1999; accepted 24 August 1999.