Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus, which naturally infects the squirrel monkey Saimiri sciureus, causing an asymptomatic but persistent infection. The latent phase of gamma-2 herpesviruses is characterized by their ability to persist in a dividing cell population while expressing a limited subset of latency-associated genes. In HVS only three genes, open reading frame 71 (ORF71), ORF72, and ORF73, are expressed from a polycistronic mRNA. ORF73 has been shown to be the only gene essential for HVS episomal maintenance and can therefore be functionally compared to the human gammaherpesvirus latency-associated proteins, EBNA-1 and Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). HVS ORF73 is the positional homologue of KSHV LANA and, although it shares limited sequence homology, has significant structural and functional similarities. Investigation of KSHV LANA has demonstrated that it is able to mediate KSHV episomal persistence by tethering the KSHV episome to host mitotic chromosomes via interactions with cellular chromosome-associated proteins. These include associations with core and linker histones, several bromodomain proteins, and the chromosome-associated proteins methyl CpG binding protein 2 (MeCP2) and DEK. Here we show that HVS ORF73 associates with MeCP2 via a 72-amino-acid domain within the ORF73 C terminus. Furthermore, we have assessed the functional significance of this interaction, using a variety of techniques including small hairpin RNA knockdown, and show that association between ORF73 and MeCP2 is essential for HVS chromosomal attachment and episomal persistence.

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-BACD71-73, 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.

Herpesviruses possess a number of characteristics which make them promising gene delivery vectors. These include their capacity to package large amounts of heterologous DNA and an ability to establish persistent, lifelong infections, where the viral genome remains as a circular non-integrated episome. Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus and is currently being developed as a potential gene delivery vector. In addition to the above properties, HVS-based vectors have the ability to infect a wide range of human cell lines and primary cultures with high efficiencies. Moreover, upon infection the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny cells upon division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained heterologous gene expression. As such, it offers the characteristics of an artificial chromosome combined with a highly efficient delivery system. This review aims to describe the assessment of HVS-based vectors in both in vitro and in vivo studies, highlighting new developments and possible applications for the treatment of genetic diseases.

Herpesvirus saimiri (HVS) establishes a persistent infection in which the viral genome persists as a circular non-integrated episome. ORF73 tethers HVS episomes to host mitotic chromosomes, allowing episomal persistence via an interaction with the chromosome-associated protein, MeCP2. Here we demonstrate that ORF73 also interacts with the linker histone H1 via its C terminus, suggesting it associates with multiple chromosome-associated proteins. In addition, we show that the C terminus is also required for the ability of ORF73 to bind the terminal repeat region of the HVS genome. These results suggest that the ORF73 C terminus contains all the necessary elements required for HVS episomal persistence. Using a range of ORF73 C terminus deletions to rescue the episomal maintenance properties of a HVSD73 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.Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesviruses which causes an asymptomatic, but persistent, infection in squirrel monkeys (Fickenscher & Fleckenstein, 2001). 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 nonintegrated 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 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;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 ...