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

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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.

mentioning

confidence: 99%

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

Griffiths

Whitehouse

2007

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“…This association is mediated by ORF73, a LANA homologue, which binds to the HVS terminal repeats and interacts with mitotic chromosomes through its C terminus (10,11,52). The similarities between LANA and ORF73 prompted Griffiths and Whitehouse to investigate the ability of MeCP2 and DEK to mediate HVS episome maintenance.…”

mentioning

confidence: 99%

The EBNA1 Protein of Epstein-Barr Virus Functionally Interacts with Brd4

Lin

Wang

Nguyen

et al. 2008

The EBNA1 protein of Epstein-Barr virus (EBV) is essential for EBV latent infection in ensuring the replication and stable segregation of the EBV genomes and in activating the transcription of other EBV latency genes. We have tested the ability of four host proteins (Brd2, Brd4, DEK, and MeCP2) implicated in the segregation of papillomavirus and Kaposi's sarcoma-associated herpesvirus to support EBNA1-mediated segregation of EBV-based plasmids in Saccharomyces cerevisiae. We found that Brd4 enabled EBNA1-mediated segregation while Brd2 and MeCP2 had a general stimulatory effect on plasmid maintenance. EBNA1 interacted with Brd4 in both yeast and human cells through N-terminal sequences previously shown to mediate transcriptional activation but not segregation. In keeping with this interaction site, silencing of Brd4 in human cells decreased transcriptional activation by EBNA1 but not the mitotic chromosome attachment of EBNA1 that is required for segregation. In addition, Brd4 was found to be preferentially localized to the FR enhancer element regulated by EBNA1, over other EBV sequences, in latently EBV-infected cells. The results indicate that EBNA1 can functionally interact with Brd4 in native and heterologous systems and that this interaction facilitates transcriptional activation by EBNA1 from the FR element.As part of their life cycle, gammaherpesviruses and papillomaviruses establish persistent infections in proliferating cells in which their double-stranded circular DNA genomes are maintained at a constant copy number. Maintenance of copy number involves the doubling of the population of viral genomes each cell cycle and a segregation mechanism to ensure equal delivery of the genomes to the daughter cells during cell division. The mechanism of mitotic segregation is conserved in the gammaherpesviruses and papillomaviruses in that, in all cases, the viral genomes are tethered to the host mitotic chromosomes through the viral origin DNA binding protein, which binds directly to the viral segregation element and interacts with one or more host chromosomal proteins.The mechanism of papillomavirus genome segregation has been studied most extensively using the bovine papillomavirus (BPV). The BPV E2 protein tethers the viral genomes to host chromosomes through interactions with multiple E2 recognition sites in the minichromosome maintenance element (MME) (22,30,38,47). E2 binds the MME through its DNA binding and dimerization domain and interacts with mitotic chromosomes through the domain responsible for transcriptional activation (6, 47). The mechanism by which the E2 transactivation domain contacts mitotic chromosomes to mediate segregation has been the subject of several studies, and considerable evidence has implicated an interaction with the bromodomain protein Brd4 in this process. Brd4 was identified as a binding partner of E2 that colocalized with E2 on host mitotic chromosomes and was shown to enable E2 to maintain plasmids containing the MME in budding yeast (Saccharomyces cerevisiae) (9,34,62). Inte...

“…Moreover, upon infection, the viral genome persists as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division, a process that is mediated by the ORF73 episomal maintenance protein (22)(23)(24)(25). This not only reduces the risk of insertional mutagenesis and gene silencing by epigenetic mechanisms but also allows the HVS-based vector to stably transduce nondividing and dividing cell populations and provide sustained heterologous gene expression.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…As HVS episomal maintenance relies on the expression of the ORF73 protein alone (22)(23)(24), modified HVS-based vectors could be produced to regulate the expression of the ORF73 protein, allowing removal of the viral episome upon successful reprogramming. This would completely eradicate the exogenous expression of reprogramming transgenes and viral DNA upon iPSC generation, a trait that is preferable to inducible integrating vectors, where there is a potential for "leaky" repression of transgenes and vector DNA remains in reprogrammed cells.…”

mentioning

confidence: 99%

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

Brown

Unger

Whitehouse

2013

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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.