Herpesvirus Saimiri Episomal Persistence Is Maintained via Interaction between Open Reading Frame 73 and the Cellular Chromosome-Associated Protein MeCP2

Griffiths, Rhoswyn; Whitehouse, Adrian

doi:10.1128/jvi.02171-06

Cited by 39 publications

(39 citation statements)

References 47 publications

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“…The viral genome exists as a stable circular episome separate from host DNA, thereby reducing the risk of insertional mutagenesis and gene silencing by epigenetic mechanisms. HVS can also persist in dividing cells, as the episomal DNA is able to replicate during latency and is transferred to daughter cells upon cell division via the open reading frame 73 (ORF73) episomal maintenance protein (22)(23)(24)(25). The virus has also been shown to efficiently infect and persist in three-dimensional multicellular spheroid cultures, a three-dimensional cell culture system that closely resembles a tumor, in addition to tumor xenografts in vivo (26)(27)(28).…”

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confidence: 99%

Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

Brown

Unger

Whitehouse

2013

J Virol

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cHerpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection, the viral genome can persist as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover, the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together, these properties are advantageous for induced pluripotent stem cell (iPSC) technology, whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4, Nanog, and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically, the putative iPCs had a number of embryonic stem cell characteristics, staining positive for alkaline phosphatase and SSEA4, in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However, differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage, they do not exhibit pluripotency. Therefore, they are hereby termed induced multipotent cancer cells.

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confidence: 99%

Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

Brown

Unger

Whitehouse

2013

J Virol

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“…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.…”

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“…293T cells were cotransfected 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).…”

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“…1a). To confirm this interaction GST pull-down assays were performed using a recombinant ORF73 protein, pET21b-73C, which expresses the ORF73 C terminus (aa 241-407) as a histidine-tagged fusion protein (Griffiths & Whitehouse, 2007). pET21b-73C was transformed and grown in BL21 Escherichia coli and subsequently expressed and bound to nickel-conjugated agarose beads.…”

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“…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 pEGFPHistoneH1 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.…”

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Mapping the minimal regions within the ORF73 protein required for herpesvirus saimiri episomal persistence

Griffiths

Harrison

Macnab

et al. 2008

Journal of General Virology

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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 ...

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Herpesvirus saimiri‐based endothelin‐converting enzyme‐1 shRNA expression decreases prostate cancer cell invasion and migration

Macnab

Lambert

et al. 2010

Intl Journal of Cancer

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The zinc metalloprotease, endothelin-converting enzyme-1 (ECE-1), which converts the mitogenic peptide endothelin-1 (ET-1) from its biologically inactive precursor big-ET-1, is commonly upregulated in prostate cancer (PC) cells. Consequently, we have sought to suppress ECE-1 expression by using RNAi as a potentially novel therapeutic approach. Therefore, a synthetic 64-nt short-hairpin RNA (shRNA), designed to target the ECE-1 gene, was expressed in an Herpesvirus saimiri (HVS)-based delivery vector. ECE-1 expression in cells transduced with the vector was examined by real-time PCR and Western blotting. The effects of ECE-1 knockdown on PC cell migration and invasion were studied using a scratch assay and Matrigel invasion. These studies, in vitro and ex vivo, demonstrated that the HVS-shRNA viruses could infect and silence ECE-1 expression effectively in human PC cells. Furthermore, it was observed that ECE-1 knockdown in either stromal cells or epithelial cells could significantly reduce invasion of PC-3 cells in coculture by 33 and 31%, respectively. In addition, suppressed migration was also observed in HVS-ECE-1 shRNA-infected PC-3 cells compared to uninfected and HVS-GFP-infected control cell cultures. These findings highlight the potential tumor-suppressing effect of ECE-1 knockdown in cancer cells and novel strategies for future therapeutic developments in advanced PC.Prostate cancer (PC) is the most frequently diagnosed cancer and the second leading cause of cancer death in western males. 1 Although androgen withdrawal can initially give effective control of cancer progression, 2 subsequent transition to the more malignant and metastatic androgen-independent form, which is neuropeptide-driven, requires novel approaches to treatment. In particular, the upregulation of protein components involved in the endothelin-1 (ET-1) signaling axis plays a key role in progression of many types of cancer, including ovarian, colorectal, breast and PC. 3-6 Specifically, plasma levels of ET-1 are abnormally elevated in patients with advanced, androgen-independent PC 7 , and the potent ET receptor subtype A (ET A R) antagonist atrasentan (ABT-627) has been reported to suppress partially prostate tumor growth. 8 Hence, inhibition of ET formation or downstream signaling could provide a novel strategy to treat advanced PC.The final and rate-limiting step in the biosynthesis of ET-1 is catalyzed by endothelin-converting enzyme-1 (ECE-1), a 120-130 kDa integral membrane-associated glycoprotein, belonging to the M13 zinc-metalloprotease family, which also includes neprilysin (NEP), the major ET-1 degradative enzyme. 9 Four isoforms of ECE-1 (ECE-1a-1d) have been identified, which are transcribed from a single gene using alternative promoters. 10,11 They have a common catalytic domain but show distinct subcellular localization because of the differences in their N-terminal tails, with ECE-1a and ECE-1c predominantly being expressed at the plasma membrane, whereas ECE-1b and ECE-1d mainly occur in endosomal membranes. 10,12 Alth...

show abstract

Herpesvirus Saimiri Episomal Persistence Is Maintained via Interaction between Open Reading Frame 73 and the Cellular Chromosome-Associated Protein MeCP2

Cited by 39 publications

References 47 publications

Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

Mapping the minimal regions within the ORF73 protein required for herpesvirus saimiri episomal persistence

Herpesvirus saimiri‐based endothelin‐converting enzyme‐1 shRNA expression decreases prostate cancer cell invasion and migration

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