Open reading frame 73 is required for herpesvirus saimiri A11-S4 episomal persistence

Michael Calderwood1,{dagger},{ddagger}, Robert E. White1,{dagger},§, Rhoswyn A. Griffiths1 and Adrian Whitehouse1,2

1 Research Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK
2 Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

Correspondence
Adrian Whitehouse
a.whitehouse{at}leeds.ac.uk


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Herpesvirus saimiri (HVS) establishes a latent infection in which the viral genome persists as a non-integrated episome. Analysis has shown that only open reading frames (ORFs) 71–73 are transcribed in an in vitro model of HVS latency. ORF73 also colocalizes with HVS genomic DNA on host mitotic chromosomes and maintains the stability of HVS terminal-repeat-containing plasmids. However, it is not known whether ORF73 is the only HVS-encoded protein required for episomal maintenance. In this study, the elements required for episomal maintenance in the context of a full-length HVS genome were examined by mutational analysis. A recombinant virus, HVS-BAC{Delta}71-73, lacking the latency-associated genes was unable to persist in a dividing cell population. However, retrofitting an ORF73 expression cassette into the recombinant virus rescued episomal maintenance. This indicates that ORF73 is the key trans-acting factor for episomal persistence and efficient establishment of a latent infection.

{dagger}These authors contributed equally to this work.

{ddagger}Present address: The Channing Laboratory, Brigham and Women's Hospital and Departments of Medicine, Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA.

§Present address: Imperial College School of Medicine, St Mary's Hospital Campus, Norfolk Place, London W2 1PG, UK.


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Herpesvirus saimiri (HVS) is the prototype gamma-2-herpesvirus, or rhadinovirus, and has significant homology with other gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein–Barr virus (EBV) and murine gammaherpesvirus-68 (MHV-68) (Albrecht et al., 1992; Russo et al., 1996; Virgin et al., 1997). In a manner comparable with that of other gammaherpesviruses, HVS establishes a latent persistent infection in lymphoid cell populations, in which the viral genome persists as a non-integrated circular episome (Fickenscher & Fleckenstein, 2001). To sustain a latent infection, the viral episomes are replicated during mitosis and efficiently segregated into daughter cells. Analysis of HVS latent gene expression has identified a cluster of genes, namely open reading frames (ORFs) 71, 72 and 73, that are transcribed as a polycistronic mRNA from a common latency-associated promoter in an in vitro model of HVS latency (Hall et al., 2000a). Similar profiles have been observed in various KSHV latently infected cell lines (Dittmer et al., 1998; Talbot et al., 1999). In both HVS and KSHV, ORF71 and ORF72 encode an anti-apoptotic FLICE-inhibitory protein (Thome et al., 1997) and a cyclin D homologue (Chang et al., 1996; Jung et al., 1994), respectively. However, ORF73 encodes a protein with no known cellular homologue.

KSHV ORF73 encodes the latency-associated nuclear antigen (LANA) (Kedes et al., 1997; Kellam et al., 1997; Rainbow et al., 1997). HVS ORF73 has only limited sequence homology with KSHV ORF73; however, both proteins share a number of common features. Both consist of a large central acidic repeat domain, flanked by a small N-terminal region and a larger C terminus (Hall et al., 2000b). A major function of LANA is to maintain the extrachromosomal viral genome during KSHV latent infection (Ballestas et al., 1999; Cotter & Robertson, 1999; Ye et al., 2004). LANA binds directly to the terminal repeats (TRs) within the KSHV genome and is sufficient for the replication and maintenance of TR-containing plasmids, tethering them to host-cell chromosomes (Ballestas et al., 1999; Cotter & Robertson, 1999; Hu et al., 2002). LANA specifically associates with host-cell mitotic chromosomes via chromosome-binding domains within its N (Piolot et al., 2001) and C termini (Ballestas et al., 1999). These domains have been shown to interact with the cellular chromosome-associated proteins histone H1, methyl-CpG-binding protein 2 and DEK (Cotter & Robertson, 1999; Krithivas et al., 2002; Shinohara et al., 2002). This suggests that KSHV ORF73 is functionally analogous to the EBV nuclear antigen 1 (EBNA1) (Kedes et al., 1997; Leight & Sugden, 2000). It has been well established that EBNA1 is the only viral protein required for EBV viral maintenance (Lee et al., 1999; Leight & Sugden, 2000; Lupton & Levine, 1985). EBNA1 binds to the family repeat element within the EBV genome (Yates et al., 1984, 1985) and interacts with metaphase chromosomes via human EBNA1 binding protein 2 (EBP2) (Kapoor & Frappier, 2003; Kapoor et al., 2001; Shire et al., 1999; Wu et al., 2002).

To date, it has been demonstrated that HVS ORF73 colocalizes with HVS genomic DNA on host mitotic chromosomes (Calderwood et al., 2004; Verma & Robertson, 2003), and maintains the stability of HVS TR-containing plasmids (Collins et al., 2002; Collins & Medveczky, 2002; Verma & Robertson, 2003). These results suggest that HVS ORF73 is a functional homolog of KSHV LANA, and provides evidence of its role in episomal maintenance. However, these results were obtained with plasmid constructs and have not conclusively determined whether HVS ORF73 is the only HVS-encoded protein required for episomal maintenance.

In this report we have utilized a HVS bacterial artificial chromosome (BAC) (White et al., 2003) to examine extensively the elements within HVS required for episomal maintenance. This virus lacks any viral transforming functions and as such will not interfere with the persistence experiments. As mentioned above, previous work has shown that only ORFs 71, 72 and 73 are expressed in an in vitro model of HVS latency, in which the HVS genome persists as a non-integrated episome (Hall et al., 2000a). These genes are transcribed as a polycistronic mRNA species from a common promoter upstream of ORF73. Therefore, to analyse the specific role of ORFs 71–73 in episomal persistence, we constructed a recombinant virus lacking ORFs 71–73, namely HVS-BAC{Delta}71-73.

The deletion of the latent transcript, encompassing ORFs 71–73, was achieved by a targeted RecA-mediated recombination system described previously (Lalioti & Heath, 2001). In order to disrupt the latent transcript, a stop codon was introduced immediately after the translation initiation site of ORF73, removing the rest of the ORF 71–73 coding region. A recombination plasmid was constructed, pKOV{Delta}71-73, which contained the ORF73 promoter and the region immediately after the ORF71 stop codon as recombination targets. The ORF73 promoter (positions 107 227 to 107 872) amplified by PCR (primers 5'-CCCGGATCCACATATATGAATGCTAGTGCAC-3' and 5'-CACACGCTAGCGCGCCATCTATAATTGCAACAAAC-3') and the region containing the latent transcript polyadenylation site (positions 104 320 to 104 702) amplified by PCR (primers 5'-CCCACCGGTGATGACTGCATTATGAAGCTC-3' and 5'-GGCAGATCTAGATAGGGCGAAGTGACCTTGC-3') were cloned together in pBSKSII+, and this targeting region was subcloned into pKOV-Kan-{Delta}Cm to construct pKOV{Delta}71-73. These were used to construct the recombinant HVS-BAC{Delta}71-73 using the protocols described previously (White et al., 2003). Following resolution of the co-integrant virus recombinants, HVS-BAC{Delta}71-73 and its revertant, HVS-BAC71-73REV, were isolated and characterized by PCR (data not shown), restriction digestion and pulsed-field gel electrophoresis (PFGE) (Fig. 1a). Furthermore, to confirm the removal of ORFs 71–73, wild-type HVS-BAC, HVS-BAC{Delta}71-73 and HVS-BAC71-73REV were transfected into the fully permissive owl monkey kidney (OMK) cell line. All three produced characteristic viral plaques; however, HVS-BAC{Delta}71-73 appeared generally smaller (data not shown). RNA was then extracted from these infected cell cultures and used in RT-PCR to examine the expression of ORFs 57, 71, 72 and 73. Results demonstrated that HVS-BAC{Delta}71-73 did not express ORFs 71–73; in contrast, the revertant showed the same transcript pattern as its parental BAC. ORF57, an immediate early viral gene, was transcribed as normal from both revertant and recombinant viruses (Fig. 1b).



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Fig. 1. Analysis of the HVS-BAC recombinant viruses. (a) To confirm the integrity of the recombinant viruses, restriction digests of wild-type HVS, HVS-BAC{Delta}71-73, HVS-BAC71-73REV and HVS-BAC{Delta}71-72 were performed using EcoRV and XhoI restriction enzymes and analysed using PFGE. (b) RT-PCR of HVS recombinants to demonstrate the lack of expression of ORFs 71–73 in HVS-BAC{Delta}71-73. (c) Colony-forming assays of HVS-BAC, HVS-BAC{Delta}71-73 and HVS-BAC71-73REV in SW480 cells. The variation between three replicate assays is indicated.

 
To test the effect of deleting ORFs 71–73 on HVS episomal persistence, viral supernatants were collected following the lytic infection of OMK cells and used to infect SW480 cells. This cell line has been previously shown to support a latent infection of wild-type HVS (Smith et al., 2001). After 24 h, cells were plated into 96-well microtitre plates and grown under hygromycin selection (200 µg ml–1) for 2 weeks. Hygromycin-resistant colonies should only be present if the HVS recombinant virus containing the hygromycin resistance gene can be maintained by episomal persistence or integration. Fig. 1(c) shows that only wild-type HVS-BAC and HVS-BAC71-73REV were capable of colony formation in a significant number of wells (94 and 89 %, respectively). The low number of positive wells (<10 %) seen for the other constructs was similar to that observed for transfected vector alone, and probably corresponds to infrequent integration events or spontaneous resistant colonies. These results suggest that ORFs 71–73 are required for episomal persistence of the HVS genome.

To confirm that these resistant colonies were due to the episomal maintenance of wild-type HVS-BAC and HVS-BAC71-73REV genomes, episome rescue assays were performed. Circular episomes were recovered from 1x106 cells by preparation of low-molecular-mass DNA, as described previously (White et al., 2003). DNA (1 µl) was then electroporated into Electromax Escherichia coli DH10B (Invitrogen) and plated on LB agar supplemented with 12·5 µg chloramphenicol ml–1. Results demonstrated that episomal DNA was recovered from resistant colonies arising from the wild-type HVS-BAC and HVS-BAC71-73REV constructs. In contrast, no bacterial colonies could be isolated from the small number of resistant colonies formed from HVS-BAC{Delta}71-73 (data not shown), further suggesting that ORFs 71–73 are required for episomal persistence.

Evidence from plasmid-based analysis has suggested that both KSHV and HVS C488 ORF73 proteins play a critical role in episomal persistence. This is further supported by previous data demonstrating that HVS A11 ORF73 can colocalize with host mitotic chromosomes (Calderwood et al., 2004). Therefore, we aimed to determine whether replacement of ORF73 alone into HVS-BAC{Delta}71-73 would rescue HVS episomal persistence. To this end, an ORF73 expression cassette was constructed containing the latent transcript polyadenylation site fused to the end of the HindIII restriction fragment containing the ORF73 gene and its promoter (positions 105 759 to 108 067 of the published sequence). This expression cassette was then subcloned into HVS-BAC{Delta}71-73 using the unique I-Ppo I restriction site previously inserted into the wild-type HVS-BAC (White et al., 2003) to produce HVS-BAC{Delta}71-72. Restriction analysis and PFGE were performed to confirm the integrity of the recombinant virus (Fig. 1a). Moreover, to demonstrate that the retrofitting of the ORF73 cassette resulted in ORF73 expression, the wild-type and recombinant viruses were used to infect OMK cells and RT-PCR was performed. The results demonstrated that HVS-BAC{Delta}71-72 did express ORF73 at similar levels to those of the wild-type virus (Fig. 2a).



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Fig. 2. ORF73 can rescue HVS episomal persistence. (a) RT-PCR of HVS recombinants to demonstrate the retrofitting of ORF73 in HVS-BAC{Delta}71-72. (b) Colony-forming assays of HVS-BAC, HVS-BAC{Delta}71-73 and HVS-BAC{Delta}71-72 in SW480 cells. The variation between three replicate assays is indicated.

 
To examine the effect on HVS episomal persistence of replacing only ORF73, HVS-BAC, HVS-BAC{Delta}71-73 and HVS-BAC{Delta}71-72 were used to infect SW480 cells. After 24 h, cells were plated into 96-well microtitre plates and grown under hygromycin selection for 2 weeks. The results demonstrated that wild-type HVS-BAC and HVS-BAC{Delta}71-72 were both capable of colony formation to levels similar to those previously seen with the wild-type HVS-BAC (Fig. 2b). Morover, episome rescue assays demonstrated that episomal DNA was recovered from HVS-BAC and HVS-BAC{Delta}71-72, in contrast to HVS-BAC{Delta}71-73 (data not shown). This suggests that ORF73 efficiently complements the inability of HVS-BAC{Delta}71-73 to establish episomal colonies and indicates that ORF73 is the key trans-acting factor for episomal persistence and for efficient establishment of a latent infection.

The gamma-2 herpesvirus ORF73 proteins have been extensively studied in vitro, and analysis has suggested a crucial role in episomal maintenance. HVS ORF73 has been shown to be expressed in an in vitro model of HVS latency (Hall et al., 2000a), to colocalize with host mitotic chromosomes (Calderwood et al., 2004; Verma & Robertson, 2003), and also to maintain the stability of HVS TR-containing plasmids (Collins et al., 2002; Collins & Medveczky, 2002; Verma & Robertson, 2003), supporting this hypothesis. In this study, we have generated a recombinant virus that disrupts the HVS latency-associated genes. Upon delivery of this virus into SW480 cells, we have demonstrated that ORFs 71–73 are required for establishment and long-term persistence of the HVS episomes. Moreover, retrofitting ORF73 alone rescued the ability of HVS-BAC{Delta}71-73 to persist as an episome, demonstrating that ORF73 is essential for the maintenance of the HVS episome in mammalian cells. We have also previously shown that a recombinant HVS lacking the TRs is unable to persist in a dividing cell population (White et al., 2003). Taken together, this data demonstrates conclusively that HVS ORF73 tethers the viral genome via the TRs to host mitotic chromosomes. The gamma-2 herpesvirus ORF73 proteins have been shown to interact with a number of cellular chromosome-associated proteins, namely histone H1, methyl-CpG-binding protein 2 and DEK (Cotter & Robertson, 1999; Krithivas et al., 2002; Shinohara et al., 2002). It will now be of interest to determine whether the HVS ORF73 protein interacts directly with these cellular proteins and whether these interactions are required for HVS episomal persistence.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants to A. W. from Yorkshire Cancer Research, Candlelighter's Trust and the Biotechnology and Biological Sciences Research Council (BBSRC).


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Received 2 June 2005; accepted 12 July 2005.



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