The Insulator Protein CTCF Binding Sites in the
            <i>orf73</i>
            /LANA Promoter Region of Herpesvirus Saimiri Are Involved in Conferring Episomal Stability in Latently Infected Human T Cells

Zielke, Katrin; Full, Florian; Teufert, Natascha; Schmidt, Monika; Müller-Fleckenstein, Ingrid; Alberter, Barbara; Ensser, Armin

doi:10.1128/jvi.06295-11

Cited by 10 publications

(9 citation statements)

References 56 publications

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“…Additionally, the IR bears a predicted, consensus binding site for the DNA-binding protein CTCF (CCCTC-binding factor), a well-known, ubiquitously expressed nuclear protein, highly conserved from Drosophila to mouse and man. CTCF is prominently involved in the three-dimensional organization of chromatin and a key player in transcriptional regulation in vertebrates37, while it appears to possess strong insulating activity in vitro 38. Importantly, it has been found to confer episomal stability to herpesvirus genomes in latently infected human T cells38.…”

Section: Discussionmentioning

confidence: 99%

The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells

Stavrou

Lazaris

Giannakopoulos

et al. 2017

Sci Rep

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Specific human chromosomal elements enhance the performance of episomal gene-transfer vectors. S/MAR-based episomal vector pEPI-eGFP transfects CD34 + haematopoietic cells, but only transiently.To address this issue we reinforced (1) transgene transcription by replacing the CMV promoter driving eGFP with the EF1/HTLV or SFFV promoters to produce vectors pEPI-EF1/HTLV and pEPI-SFFV, respectively; and (2) plasmid replication by inserting the replication-Initiation Region (IR) from the β-globin locus into vector pEPI-SFFV to produce vector pEP-IR. All vectors supported stable transfections in K562 cells. Transfections of CD34 + cells from peripheral blood of healthy donors reached 30% efficiency. Upon evaluation of CD34 + /eGFP + cells in colony-forming cell (CFC) assays, vector pEP-IR showed superior performance after 14 days, by fluorescent microscopy: 100% eGFP + -colonies against 0% for pEPI-eGFP, 56.9% for pEPI-SFFV and 49.8% for pEPI-EF1/HTLV; 50% more plasmid copies per cell and 3-fold eGFP expression compared to the latter two constructs, by quantitative (q)PCR and RT-qPCR, respectively. Importantly, the establishment rate in CFC assays was 15% for pEP-IR against 5.5% for pEPI-SFFV and 5% for pEPI-EF1/HTLV. Vector pEP-IR shows extremely low delivery rate but supports eGFP expression in thalassaemic mouse haematopoietic progenitor cells. The IR is a novel human control element for improved episomal gene transfer into progenitor cells.The design and use of extrachromosomal vectors, suitable for efficient and stable transfection of haematopoietic progenitor cells, is an important goal for the gene therapy of haemoglobinopathies. The development of extrachromosomal vectors has mainly been driven by the need to address the safety issue of gene therapy vectors, in particular, the problem of insertional mutagenesis 1 , and involves vectors such as self-replicating stable episomes 2 , pFARs-plasmids free of antibiotic resistance markers 3 , and minicircle DNA plasmid derivatives lacking a bacterial backbone 4 . The presence of the scaffold/matrix attachment region (S/MAR) also confers long-term mitotic stability to integration-deficient lentiviral, episomal vectors 5,6 ; however, that of the truncated S/MAR does not improve episomal retention 7 .Key issues in the development of episomal vectors are currently the establishment in the host nucleus 8,9 , the transgene expression 10,11 and the delivery in progenitor cells 10 . The prototype episomal vector pEPI-1 2 does not code for any viral protein, and it contains the S/MAR from the 5′ end of the human β -interferon gene 2 , an element that facilitates the vector's nuclear retention.The S/MARs are AT rich chromosomal elements that play a role in chromatin boundary formation 12 and bind to SAF-A protein 13 , mediating the tethering of pEPI-1 plasmid to the nuclear matrix. A prerequisite for

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Section: Discussionmentioning

confidence: 99%

The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells

Stavrou

Lazaris

Giannakopoulos

et al. 2017

Sci Rep

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“…Primary rhesus fibroblasts (kindly provided by A. Kaur, Harvard Medical School, Southborough, MA), OMK cells, and Vero cells were cultivated in Dulbecco's minimal essential medium (Gibco/BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum and gentamicin. The HVS strain employed in this study was obtained by reconstitution of infectious viruses using Bac43MOD containing the genomic sequence of HVS strain C488 (10) and a cytomegalovirus (CMV) immediate-early promoter-driven enhanced green fluorescent protein (EGFP) expression cassette inserted in the SwaI site at position 5656, analogous to earlier descriptions (42,47). Stocks of wild-type (wt) and recombinant viruses were prepared, and the amount of viral genomes in the supernatant of infected OMK cells was quantified by quantitative PCR of the MCP locus with primers MCP_for (5=-CCATTTGCCTGTGTTGAGAGTTAA-3=) and MCP_rev (5=-CTCATTACCAGACCCATGTTATGAA-3) and probe MCP_probe (5=/56-FAM/CTCCGAGAG/ZEN/AGCCTATCTGAGATG CCC/3lABkFQ/-3=, where FAM is 6-carboxyfluorescein, ZEN is internal ZEN Quencher, and 3IABKFQ is 3= Ioua Black FQ Quencher) (Integrated DNA Technologies, Coralville, IA).…”

Section: Cells and Virusesmentioning

confidence: 99%

Herpesvirus Saimiri Antagonizes Nuclear Domain 10-Instituted Intrinsic Immunity via an ORF3-Mediated Selective Degradation of Cellular Protein Sp100

Full

Reuter

Zielke

et al. 2012

J Virol

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In recent studies, the nuclear domain 10 (ND10) components PML, Sp100, human Daxx (hDaxx), and ATRX were identified to be cellular restriction factors that are able to inhibit the replication of several herpesviruses. The antiviral function of ND10, however, is antagonized by viral effector proteins by a variety of strategies, including degradation of PML or relocalization of ND10 proteins. In this study, we analyzed the interplay between infection with herpesvirus saimiri (HVS), the prototypic rhadinovirus, and cellular defense by ND10. In contrast to other herpesviruses, we found that HVS specifically degraded the cellular ND10 component Sp100, whereas other factors like PML or hDaxx remained intact. We could further identify the ORF3 tegument protein of HVS, which shares homology with the cellular formylglycinamide ribotide amidotransferase (FGARAT) enzyme, to be the viral factor that induces the proteasomal degradation of Sp100. Interestingly, recent studies showed that the ORF3-homologous proteins ORF75c of murine gammaherpesvirus 68 and BNRF-1 of Epstein-Barr virus modulate the ND10 proteins PML and ATRX, respectively, suggesting that the ND10 targets of viral FGARAT-homologous proteins diversified during evolution. Furthermore, a virus with the ORF3 deletion was efficiently complemented in Sp100-depleted cells, indicating that Sp100 is able to inhibit HVS in the absence of antagonistic mechanisms. In contrast, we observed that PML, which was neither degraded nor redistributed after HVS infection, strongly restricted both wild-type HVS and virus with the ORF3 deletion. Thus, HVS may lack a factor that efficiently counteracts the repressive function of PML, which may foster latency as the outcome of infection.

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“…Herpesviruses are characterized by their ability to establish lifelong latency in the host. Only limited numbers of viral genes are expressed during latent infection, while multiple copies of the viral genome persist as circular episomes within the nuclei of host cells (Zhou & Gao 2011;Zielke et al 2012). Our PCR results indicated that the left and right TRs of the CyHV-3 genome were fused in a head-to-tail manner followed by sequence rearrangements by temperature upshift from 25°C to 35°C.…”

Section: Influence Of Temperature Shift On Cyhv-3 Replicationmentioning

confidence: 79%

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency ofCyHV‐3 by temperature shift in the cells

Imajoh

Fujioka

Furusawa

et al. 2014

Journal of Fish Diseases

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A new cell line named CCF-K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV-3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV-3-infected CCF-K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV-3 was produced stably in CCF-K104 cells over 30 viral passages. Our findings showed that CCF-K104 is a useful cell line for isolation and productive replication of CyHV-3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real-time PCR showed that CyHV-3 was present with low viral DNA loads, suggesting that CyHV-3 may establish latent infection in CCF-K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV-3 genome arranged in a head-to-tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV-3.

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The Insulator Protein CTCF Binding Sites in the orf73 /LANA Promoter Region of Herpesvirus Saimiri Are Involved in Conferring Episomal Stability in Latently Infected Human T Cells

Cited by 10 publications

References 56 publications

The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells

The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells

Herpesvirus Saimiri Antagonizes Nuclear Domain 10-Instituted Intrinsic Immunity via an ORF3-Mediated Selective Degradation of Cellular Protein Sp100

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency ofCyHV‐3 by temperature shift in the cells

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