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Short Communication |
1 Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK
2 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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ABSTRACT |
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73 recombinant virus, we show that a C terminus region comprising residues 285–407 is sufficient to maintain the HVS episome in a dividing cell population. |
MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). In a manner comparable to other gamma herpesviruses, HVS establishes a latent persistent infection in lymphoid and epithelial cell populations, where the viral genome persists as a high copy number circular non-integrated episome (Kaschka-Dierich et al., 1982; Werner et al., 1977). This ability to persist as an non-integrated episome has led to the development of HVS as a possible gene delivery vector (Calderwood et al., 2004b; Griffiths et al., 2006). To sustain this persistent infection in a dividing cell population, the viral episomes must be replicated during mitosis and segregated efficiently into daughter cells (Biesinger et al., 1992). The HVS ORF73 protein has been implicated in the maintenance of the viral episome during cell division (Hall et al., 2000a). HVS ORF73 colocalizes with HVS genomic DNA on host mitotic chromosomes (Calderwood et al., 2004a; Verma & Robertson, 2003), and it maintains the stability of HVS terminal repeat (TR)-containing plasmids (Collins et al., 2002; Collins & Medveczky, 2002; Verma & Robertson, 2003). Moreover, deletion analysis of the HVS-BAC demonstrated that both ORF73 and the TR regions of HVS are required for episomal maintenance (Calderwood et al., 2005; Collins et al., 2002; White et al., 2003). These results suggest that the HVS ORF73 is a functional homologue of Kaposi's sarcoma associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA; Ballestas et al., 1999; Ballestas & Kaye, 2001), and tethers the HVS episomes to host mitotic chromosomes, via the TR region of the HVS genome.
Studies of KSHV LANA have shown that chromosome binding is achieved through interactions with multiple cellular chromosome-associated proteins, including MeCP2, DEK, core histones H2A and H2B, the linker histone H1 and cellular bromodomain-containing proteins (Barbera et al., 2006; Cotter & Robertson, 1999; Krithivas et al., 2002; Shinohara et al., 2002; Viejo-Borbolla et al., 2005). We have recently demonstrated that HVS ORF73 associates with MeCP2 and shown that this association is essential for HVS chromosomal attachment and episomal persistence (Griffiths & Whitehouse, 2007). In this report, we have investigated whether HVS ORF73 interacts with other chromosome-associated proteins to ensure mitotic chromosome association. We demonstrate that the HVS ORF73 C terminus interacts with the linker histone H1. In addition, we demonstrate that the C terminus is required for the ability of ORF73 to bind the TR region of the HVS genome. These results suggest that the ORF73 C terminus contains all the necessary elements required for HVS episomal persistence. Therefore, we utilized a range of ORF73 C terminus deletions to identify the minimal region with the ORF73 C terminus able to rescue the episomal maintenance properties of a HVS73 recombinant virus. Results show that a C terminus region comprising residues 285–407 is the minimal domain sufficient to maintain the HVS episome in a dividing cell population.
To further investigate the interactions between HVS ORF73 and chromosome-associated proteins, co-immunoprecipitation assays were performed. 293T cells were co-transfected with pEGFP-HistoneH1 in the presence of pMyc or p73-myc (Calderwood et al., 2004a). To ensure that the interaction between ORF73 and HistoneH1 was not due to a common affinity for DNA, co-immunoprecipitation assays were performed with cell extracts which remained untreated or treated with DNase I, as described previously (Griffiths & Whitehouse, 2007). To confirm that DNase I treatment had been successful, untreated and treated cell extracts were used as templates within a PCR utilizing primers directed against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This analysis demonstrated that complete digestion of cellular DNA had occurred following addition of DNase I (Fig. 1a). Subsequently, the cell extracts were incubated with an ORF73-specific antibody and the immunocomplex captured using protein-A agarose. Histone H1 was then detected by Western blotting using a 1 : 2000 dilution of GFP-specific antibody (Clontech). Results show that ORF73 interacted with HistoneH1 and that this interaction was DNA-independent (Fig. 1a).
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). pET21b-73C was transformed and grown in BL21 Escherichia coli and subsequently expressed and bound to nickel-conjugated agarose beads. Histone H1–GFP or GFP proteins were expressed in 293T cells and the cell extracts incubated with the ORF73C-beads. After washing, proteins bound to the ORF73C-beads were separated by SDS-PAGE and detected by Western blotting using a GFP-specific antibody. The results confirm that ORF73C specifically bound the chromosome-associated protein HistoneH1 (Fig. 1b
). Moreover, as we have previously shown that ORF73 also interacts with MeCP2, it suggests that ORF73 can interact with multiple chromosome-associated proteins.
To further investigate this interaction, deletion analysis was used to determine the ORF73 minimal domain required to bind to histone H1. The ORF73C histidine-tagged fusion deletion series (Fig. 1c) (Griffiths & Whitehouse, 2007) was expressed as recombinant histidine-tagged proteins, bound to Ni-NTA agarose beads and incubated with a pEGFP-HistoneH1 transfected cell extract. Although several of the proteins were insoluble and could not be used in the assay, analysis of these deletion proteins demonstrated that an overlapping fragment between deletion 4 and 10 encompassing ORF73 aa 324–379 is sufficient for histone H1 binding (Fig. 1d). Interestingly, this region is similar to the domain which interacts with MeCP2, namely aa 324–396. However, it has been shown previously that the minimal region of the ORF73 C terminus for chromosomal association is aa 285–407 (Calderwood et al., 2004a), which suggests that additional domains either side of the minimal MeCP2- and histone H1-binding regions, termed chromosome association sites (CAS) 1 and 2, are required for chromosome association.
In addition to binding host mitotic chromosomes, ORF73 must bind the TR region to maintain the HVS episome in a dividing cell population. Therefore, the specific ORF73 domain responsible for binding HVS TR DNA was investigated using chromatin immunoprecipitation (ChIP) experiments. The previously constructed ORF73 deletion series (Hall et al., 2000b), consisting of pEGFP-73NC, pEGFP-73N, pEGFP-73C (Fig. 2a) or empty pEGFP vector, were co-transfected into 293T cells together with a plasmid containing four copies of the HVS TR sequence (pHVS-TR+hyg). After 24 h the cells were harvested and ChIP assays performed using the ChIP assay kit (Upstate Biotechnology). Chromatin extracts, cross-linking, sonication, immunoprecipitation, agarose bead elution and protein removal were carried out based on the manufacturer's protocol. DNA recovered from immunoprecipitates with the GFP-specific polyclonal antibody was used as a template for PCR amplifications using primers specifically directed against unique HVS TR sequences. To eliminate the possibility of non-specific binding resulting in precipitation of TR DNA, reactions were also prepared in the absence of immunoprecipitating antibody. Results shown in Fig. 2(b) demonstrate that, in the presence of the GFP-specific antibody, TR DNA was successfully immunoprecipitated in association with both EGFP–73NC and EGFP–73C protein, resulting in amplification of the expected 1444 bp DNA fragment. These results demonstrate that the ORF73 C terminus is sufficient for binding to HVS TR DNA.
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, each deletion protein includes various combinations of the previously defined ORF73C chromosome association sites (CAS1 or 2), or the histone H1-binding domain. To confirm that each vector expressed an EGFP-tagged protein of the expected molecular mass, the vectors were transfected into 293T cells and cell extracts analysed by SDS-PAGE and Western blotting. Analysis using a GFP-specific antibody indicates that each vector successfully expresses a GFP-tagged ORF73C deletion protein of the correct size (Fig. 2d). Each deletion was then assessed for its ability to bind HVS TR DNA using ChIP assays. As shown in Fig. 2(e) full-length ORF73C and EGFP–73C5 successfully bound HVS TR DNA. These results indicate that ORF73 residues 324–407 are sufficient for binding to HVS TR DNA.
The deletion analysis of ORF73 suggests that the minimal domain for both chromosome association and TR DNA binding resides in the C terminus. Therefore, we next aimed to determine which residues within the C terminus were sufficient to support HVS episomal maintenance. We have demonstrated previously that replacement of the complete ORF73 gene into a recombinant HVS lacking ORFs 71–73 is sufficient to rescue HVS episomal persistence (Calderwood et al., 2005). We therefore assessed whether any C-terminal deletion constructs could rescue the episomal persistence ability of HVS-BAC71-73. To this end, SW480 cells were transfected with each N- and C-terminal ORF73 deletion construct (Fig. 2), after 24 h the transfected cells were superinfected with HVS-BAC71-73 (m.o.i. of 1) and maintained in 600 µg G418 ml–1 and 200 µg hygromycin ml–1, which selected for the 73 deletion constructs and HVS-BAC, respectively, for a further 24 h. SW480 cells were used in this analysis as we have previously shown that HVS establishes a latent infection in these cells where the genome persists as a non-integrated episome (Smith et al., 2001). The cells were then trypsinized and diluted to a cell density of 1x102 cells ml–1. The cells were then seeded at approximately 10 cells per single well in 96-well microtitre plates and grown under G418 and hygromycin selection for 2 weeks. The plates were then analysed for colony formation and scored as a percentage of wells positive for colony outgrowth. Results demonstrated that only cells pre-transfected with pEGFP-73NC, pEGFP-73C or pEGFP-73C-CAS1+2 enabled HVS-BAC71-73 to be maintained in a dividing cell population (Fig. 3). Moreover, transfected cells were also seeded in duplicate at 1x104 cells and grown under G418 and hygromycin selection for 2 weeks. Similar results were observed as above, where cell growth was only present in cells pre-transfected with pEGFP-73NC, pEGFP-73C or pEGFP-73C-CAS1+2. RNA was then isolated from each cell line and used in RT-PCR analysis to confirm the expression of each ORF73 expression construct in SW480 cells, following 2 weeks growth in selection. Results demonstrate that expression of each ORF73 C-terminal deletion was observed in each selected cell line (Fig. 3b). To confirm the presence of the HVS-BAC71-73 episome in each selected cell line, DNA was isolated from cells after 14 days selection using the low molecular mass DNA isolation method (White et al., 2003). The DNA (1 µl) was then electroporated into E. coli ElectroMAX DH10B (Invitrogen) and plated on LB agar supplemented with 12.5 µg chloramphenicol ml–1. Results demonstrated that episomal DNA was recovered from cells pre-transfected with pEGFP-73NC, pEGFP-73C or pEGFP-73C-CAS1+2. No DNA was isolated from the few small colonies pre-transfected with the other ORF73 constructs. To confirm the bacterial colonies were due to transformation by HVS episomes, DNA isolated from these bacterial colonies was analysed by restriction digest and pulsed-field gel electrophoresis. Restriction digests demonstrate that the episomes isolated were consistent with those expected for HVS-BAC71-73 (Fig. 3c). These results therefore indicate that the ORF73 C terminus can rescue the episomal persistence ability of HVS-BAC71-73. Moreover, residues 285–407 within the ORF73 C terminus, which encompass both CAS1 and CAS2, is the minimal region required for episomal persistence and for efficient establishment of a latent infection.
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). Therefore, even ubiquitous chromosome-associated proteins such as histone H1 may remain in a constant state of chromosome association and disassociation. Hence, the ability of HVS ORF73 to bind several tightly associated chromosomal proteins provides an effective mechanism by which it can secure long-term latent persistence.
Moreover, we demonstrate that the C terminus can also bind TR DNA. Interesting, Cotter et al. demonstrated that a 200 amino acid domain within the C terminus of KSHV LANA is sufficient for binding to KSHV TR DNA (Cotter et al., 2001). Deletion analysis of ORF73C demonstrated that only the terminal 83 aa of the protein are required for interaction with the TRs. The ORF73C TR-binding domain includes the MeCP2- and histone H1-binding region and also contains the previously identified CAS2. Analysis of ORF73C multimerization has shown that CAS2 is essential for the formation of ORF73C homo-multimers (Calderwood et al., 2004a). Therefore, akin to many DNA-binding proteins, self-association of ORF73 is probably required to bind DNA.
In summary, we demonstrate the HVS ORF73 C terminus contains multiple functional domains required for chromosome association and DNA binding. Moreover, the minimal C terminus region, comprising residues 285–407, is sufficient to maintain the HVS episome in a dividing cell population. This analysis will help towards the development of safe replication-disabled HVS-based vectors for gene therapy applications, such as the HVS amplicon system (Macnab et al., 2008). The region identified herein, required for HVS episomal maintenance, will help minimize the viral sequence required in these HVS amplicon-based vectors.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 April 2008;
accepted 25 June 2008.
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