Episomal Maintenance Research Articles

The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

Abstract 465: Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors

Abstract Most humans infected with Epstein-Barr virus (EBV) remain asymptomatic throughout the whole life. However, EBV is linked to several human cancers, including Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC), and gastric carcinoma. Among these EBV-associated tumors, EBV genome is present in 100% of NPC tumor cells. In NPC cells, EBV establishes a latent infection, expressing limited number of viral proteins but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, also known as BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs includes BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). Although various functions of BART miRNAs have been identified, the role of EBV lnc-BARTs remains largely unknown. In this study, we provide evidence demonstrating that lnc-BARTs functions as a regulatory long non-coding RNA, referred to as lnc-BARTs. These lnc-BARTs play keys role in modulating the core networks that maintains EBV latency and promotes NPC oncogenesis through epigenetic mechanisms. Our study found that the apoptosis rate of NPC cells is significantly increased following the knockdown of EBV lnc-BARTs, indicating their anti-apoptotic role in EBV-associated tumors. Mass spectrometry analysis identified several host factors, including transcriptional regulators BRD4 and SC35’ interact with EBV lnc-BARTs in NPC cells. RNA-FISH assays showed lnc-BARTs co-localized with BRD4 and SC35 complexes in nuclear s peckles and such interaction were disrupted by JQ1, a BRD4 competitive inhibitor. Binding of lnc-BARTs to BRD4 RNA was confirmed using immunoprecipitation and pulldown assays. ATAC sequencing and transcriptome analyses showed that BART knockdown in EBV-harboring NPC cell line C666-1 led to reduced chromatin accessibility, downregulation of oncogenes, and decreased EBV episome replication and maintenance. Quantitative qPCR and DNA FISH analyses of cell samples treated with shRNA targeting lnc-BARTs or BRD4 reduced EBV copy numbers and EBNA1 RNA expression levels in EBV-infected NPC cells. Finally, ChIP sequencing analysis revealed that lnc-BARTs is critical for BRD4 binding to the oriP region of the EBV genome. These findings support a mechanism by which EBV lnc-BARTs epigenetically regulate host gene expression through functional interactions with elongation regulatory machinery and contribute to the maintenance of EBV latent genome by interaction with BRD4 complex. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Honglin Chen. Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 465.

Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of VK-2019, a selective EBNA1 inhibitor.

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Functional analysis of the putative centromere sequences of marine oleaginous diatom Fistulifera solaris

Trans-kingdom conjugation-based transformation with oriT-containing episomal plasmids is an emerging technique to achieve genetic engineering in diatoms. The native centromeres lacking any consensus motifs and budding yeast-derived centromere/autonomous replication sequence accompanied by a histidine synthase gene (CEN6-ARSH4-HIS3) supported the plasmid maintenance in model diatoms. However, it remained elusive whether these host-vector systems are universally applicable in industrially important non-model diatom species. In this study, we found the consensus motifs in the putative centromeres of Fistulifera solaris; some exhibited characteristic dispersion patterns along the chromosomes. Furthermore, we investigated whether (1) oriT-containing plasmids could be transferred from the Escherichia coli strain S17-1 expressing tra genes to the oleaginous diatom F. solaris, and (2) the putative centromeres of F. solaris and yeast CEN6-ARSH4-HIS3 could support the episomal maintenance in F. solaris. As a result, transformant clones were successfully obtained through a drug-registrant selection process. PCR testing further confirmed the transfer of the plasmid sequences in the diatom. Some of the centromeres containing the consensus motifs marginally contributed to the transformation; however, the differences in terms of the transformation efficiency in the presence or absence of F. solaris-derived putative centromeres and yeast-derived CEN6-ARSH4-HIS3 were not statistically significant. The episome rescue experiment did not provide evidence that the transferred plasmids were maintained as episomes. These results suggest that these centromeric sequences might not be solely responsible for episomal maintenance in F. solaris.

Regulation of EBNA1 protein stability and DNA replication activity by PLOD1 lysine hydroxylase.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

Cryo-EM Structure and Functional Studies of EBNA1 Binding to the Family of Repeats and Dyad Symmetry Elements of Epstein-Barr Virus oriP.

Epstein-Barr nuclear antigen 1 (EBNA1) is a multifunctional viral-encoded DNA-binding protein essential for Epstein-Barr virus (EBV) DNA replication and episome maintenance. EBNA1 binds to two functionally distinct elements at the viral origin of plasmid replication (oriP), termed the dyad symmetry (DS) element, required for replication initiation and the family of repeats (FR) required for episome maintenance. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the EBNA1 DNA binding domain (DBD) from amino acids (aa) 459 to 614 and its interaction with two tandem sites at the DS and FR. We found that EBNA1 induces a strong DNA bending angle in the DS, while the FR is more linear. The N-terminal arm of the DBD (aa 444 to 468) makes extensive contact with DNA as it wraps around the minor groove, with some conformational variation among EBNA1 monomers. Mutation of variable-contact residues K460 and K461 had only minor effects on DNA binding but had abrogated oriP-dependent DNA replication. We also observed that the AT-rich intervening DNA between EBNA1 binding sites in the FR can be occupied by the EBNA1 AT hook, N-terminal domain (NTD) aa 1 to 90 to form a Zn-dependent stable complex with EBNA1 DBD on a 2×FR DNA template. We propose a model showing EBNA1 DBD and NTD cobinding at the FR and suggest that this may contribute to the oligomerization of viral episomes important for maintenance during latent infection. IMPORTANCE EBV latent infection is causally linked to diverse cancers and autoimmune disorders. EBNA1 is the viral-encoded DNA binding protein required for episomal maintenance during latent infection and is consistently expressed in all EBV tumors. The interaction of EBNA1 with different genetic elements confers different viral functions, such as replication initiation at DS and chromosome tethering at FR. Here, we used cryo-EM to determine the structure of the EBNA1 DNA-binding domain (DBD) bound to two tandem sites at the DS and at the FR. We also show that the NTD of EBNA1 can interact with the AT-rich DNA sequence between tandem EBNA1 DBD binding sites in the FR. These results provide new information on the mechanism of EBNA1 DNA binding at DS and FR and suggest a higher-order oligomeric structure of EBNA1 bound to FR. Our findings have implications for targeting EBNA1 in EBV-associated disease.

Herpesvirus-induced spermidine synthesis and eIF5A hypusination for viral episomal maintenance.

Spermidine is essential for cellular growth and acts as a prerequisite of hypusination, a post-translational modification of eukaryotic initiation factor 5A (eIF5A), allowing the translation of polyproline-containing proteins. Here, we show that oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) increases spermidine synthesis and eIF5A hypusination to enhance expression of polyproline-containing latency-associated nuclear antigen (LANA) for viral episomal maintenance. KSHV upregulates intracellular spermidine levels by dysregulating polyamine metabolic pathways in three-dimensional (3D) culture and 2D de novo infection conditions. Increased intracellular spermidine leads to increased eIF5A hypusination, ultimately enhancing LANA expression. In contrast, inhibition of spermidine synthesis or eIF5A hypusination alleviates LANA expression, decreasing viral episomal maintenance and KSHV-infected cell proliferation invitro and invivo, which is reversed by spermidine supplement. This demonstrates that KSHV hijacks spermidine synthesis and eIF5A hypusination pathways to enhance LANA expression for viral episomal maintenance, suggesting polyamine metabolism and eIF5A hypusination as therapeutic targets for KSHV-induced tumorigenesis.

Assembly of Multiple Full-Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System.

Human artificial chromosomes (HACs) are gene delivery vectors that have been used for decades for gene functional studies. HACs have several advantages over viral‐based gene transfer systems, including stable episomal maintenance in a single copy in the cell and the ability to carry up to megabase‐sized genomic DNA segments. We have previously developed the alphoidtetO‐HAC, which has a single gene acceptor loxP site that allows insertion of an individual gene of interest using Chinese hamster ovary (CHO) hybrid cells. The HAC, along with a DNA segment of interest, can then be transferred from donor CHO cells to various recipient cells of interest via microcell‐mediated chromosome transfer (MMCT). Here, we detail a protocol for loading multiple genomic DNA segments or genes into the alphoidtetO‐HAC vector using an iterative integration system (IIS) that utilizes recombinases Cre, ΦC31, and ΦBT. This IIS‐alphoidtetO‐HAC can be used for either serially assembling genomic loci or fragments of a large gene, or for inserting multiple genes into the same artificial chromosome. The insertions are executed iteratively, whereby each round results in the insertion of a new DNA segment of interest. This is accompanied by changes of expression of marker fluorescent proteins, which simplifies screening of correct clones, and changes of selection and counterselection markers, which constitutes an error‐proofing mechanism that removes mis‐incorporated DNA segments. In addition, the IIS‐alphoidtetO‐HAC carrying the genes can be eliminated from the cells, offering the possibility to compare the phenotypes of human cells with and without functional copies of the genes of interest. The resulting HAC molecules may be used to investigate biomedically relevant pathways or the regulation of multiple genes, and to potentially engineer synthetic chromosomes with a specific set of genes of interest. The IIS‐alphoidtetO‐HAC system is expected to be beneficial in creating multiple‐gene humanized models with the purpose of understanding complex multi‐gene genetic disorders. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Integration of the first DNA segment of interest into the IIS‐alphoidteto‐HAC Basic Protocol 2: Integration of a second DNA segment of interest into the IIS‐alphoidteto‐HAC Basic Protocol 3: Integration of a third DNA segment of interest into the IIS‐alphoidteto‐HAC Support Protocol: Fluorescence in situ hybridization analysis for the circular IIS‐alphoidtetO‐HAC

USING A NONVIRAL AND NONINTEGRATING DNA VECTOR TO GENERATE SAFE CAR-T CELLS

CAR-T cell therapy has proven its importance in cancer treatment and currently there are five approved products on the market. However, these products are based on viral vectors, which presents a risk of insertional mutagenesis, it can also be immunogenic and its manufacture is extremely expensive. The development of a nonviral vector, suitable for an efficient and stable transfection of NK and T cells is the goal of this study. In this study we use a special episomal vector (extrachromosomal vectors) with i) scaffold/matrix attachment region (S/MAR) motifs that can actively mediate the episomal maintenance, ii) a plasmid replication enhancer, the replication-Initiation Region (IR) from the β-globin locus (pEP-IR) and iii) a CAR anti CD19 (pEP-IR-CAR). We electroporated activated and non-activated T cells and NK cells with different plasmid concentrations. We evaluated transfection efficiency and viability. Our findings showed that unstimulated T cells are transfected more efficiently than activated T cells. Transfection of unstimulated T cells with 20 ug of supercoiled pEP-IR-GFP plasmid reached 22 to 63% of GFP and viability around 54% depending on the culture condition used after electroporation (FBS% and IL2). We then accessed the efficacy of pEP-IR-CAR and CAR expression, confirmed in 3,2% of the cells with 89% of viability. Cells expressing CAR on their surface were marked with anti-CAR antibody Biotin-SP-conjugated and selected by anti-Biotin microbeads, achieving an enrichment of 54,9% CAR+ cells. Our results showed that we established an optimized protocol resulting in an acceptable transfection efficiency and the transfected cells can be selected. So, the next step is to perform in vitro cytotoxicity and in vivo assays with the episomal CAR+ cells. Financially supported by FAPESP 2020/09206-4, FAPESP 2019/25309-0, CTC Center for Cell-based Therapy (FAPESP 2013/08135-2), and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773).

Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP invivo and for formation of SDS-resistant complexes invitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.

EXPRESSION OF E8^E2 IS REQUIRED FOR WART FORMATION BY MOUSE PAPILLOMAVIRUS 1 IN VIVO.

Human papillomavirus (HPV) E1 and E2 proteins activate genome replication. E2 also modulates viral gene expression and is involved in the segregation of viral genomes. In addition to full length E2, almost all PV share the ability to encode an E8^E2 protein, that is a fusion of E8 with the C-terminal half of E2 which mediates specific DNA-binding and dimerization. HPV E8^E2 acts as a repressor of viral gene expression and genome replication. To analyze the function of E8^E2 in vivo, we used the Mus musculus PV1 (MmuPV1)-mouse model system. Characterization of the MmuPV1 E8^E2 protein revealed that it inhibits transcription from viral promoters in the absence and presence of E1 and E2 proteins and that this is partially dependent upon the E8 domain. MmuPV1 genomes, in which the E8 ATG start codon was disrupted (E8-), displayed a 10- to 25-fold increase in viral gene expression compared to wt genomes in cultured normal mouse tail keratinocytes in short-term experiments. This suggests that the function and mechanism of E8^E2 is conserved between MmuPV1 and HPVs. Surprisingly, challenge of athymic nude Foxn1nu/nu mice with MmuPV1 E8- genomes did not induce warts on the tail in contrast to wt MmuPV1. Furthermore, viral gene expression was completely absent at E8- MmuPV1 sites 20 - 22 weeks after DNA challenge on the tail or quasivirus challenge in the vaginal vault. This reveals that expression of E8^E2 is necessary to form tumors in vivo and that this is independent from the presence of T-cells.IMPORTANCE HPV encode an E8^E2 protein which acts as repressors of viral gene expression and genome replication. In cultured normal keratinocytes, E8^E2 is essential for long-term episomal maintenance of HPV31 genomes, but not for HPV16. To understand E8^E2's role in vivo, the Mus musculus PV1 (MmuPV1)-mouse model system was used. This revealed that E8^E2's function as a repressor of viral gene expression is conserved. Surprisingly, MmuPV1 E8^E2 knock out genomes did not induce warts in T-cell deficient mice. This shows for the first time that expression of E8^E2 is necessary for tumor formation in vivo independently of T cell immunity. This indicates that E8^E2 could be an interesting target for anti-viral therapy in vivo.

Engineering of human induced pluripotent stem cells via human artificial chromosome vectors for cell therapy and disease modeling

Genetic engineering of induced pluripotent stem cells (iPSCs) holds great promise for gene and cell therapy as well as drug discovery. However, there are potential concerns regarding the safety and control of gene expression using conventional vectors such as viruses and plasmids. Although human artificial chromosome (HAC) vectors have several advantages as a gene delivery vector, including stable episomal maintenance and the ability to carry large gene inserts, the full potential of HAC transfer into iPSCs still needs to be explored. Here, we provide evidence of a HAC transfer into human iPSCs by microcell-mediated chromosome transfer via measles virus envelope proteins for various applications, including gene and cell therapy, establishment of versatile human iPSCs capable of gene loading and differentiation into T cells, and disease modeling for aneuploidy syndrome. Thus, engineering of human iPSCs via desired HAC vectors is expected to be widely applied in biomedical research.

Evidence for Tethering of Human Cytomegalovirus Genomes to Host Chromosomes.

Tethering of viral genomes to host chromosomes has been recognized in a variety of DNA and RNA viruses. It can occur during both the productive cycle and latent infection and may impact viral genomes in manifold ways including their protection, localization, transcription, replication, integration, and segregation. Tethering is typically accomplished by dedicated viral proteins that simultaneously associate with both the viral genome and cellular chromatin via nucleic acid, histone and/or non-histone protein interactions. Some of the most prominent tethering proteins have been identified in DNA viruses establishing sustained latent infections, including members of the papillomaviruses and herpesviruses. Herpesvirus particles have linear genomes that circularize in infected cell nuclei and usually persist as extrachromosomal episomes. In several γ-herpesviruses, tethering facilitates the nuclear retention and faithful segregation of viral episomes during cell division, thus contributing to persistence of these viruses in the absence of infectious particle production. However, it has not been studied whether the genomes of human Cytomegalovirus (hCMV), the prototypical β-herpesvirus, are tethered to host chromosomes. Here we provide evidence by fluorescence in situ hybridization that hCMV genomes associate with the surface of human mitotic chromosomes following infection of both non-permissive myeloid and permissive fibroblast cells. This chromosome association occurs at lower frequency in the absence of the immediate-early 1 (IE1) proteins, which bind to histones and have been implicated in the maintenance of hCMV episomes. Our findings point to a mechanism of hCMV genome maintenance through mitosis and suggest a supporting but non-essential role of IE1 in this process.

STUB1 is targeted by the SUMO-interacting motif of EBNA1 to maintain Epstein-Barr Virus latency.

Latent Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including B-cell lymphomas and epithelial tumors. EBNA1 is a key antigen expressed in all EBV-associated tumors during latency that is required for maintenance of the EBV episome DNA and the regulation of viral gene transcription. However, the mechanism utilized by EBV to maintain latent infection at the levels of posttranslational regulation remains largely unclear. Here, we report that EBNA1 contains two SUMO-interacting motifs (SIM2 and SIM3), and mutation of SIM2, but not SIM3, dramatically disrupts the EBNA1 dimerization, while SIM3 contributes to the polySUMO2 modification of EBNA1 at lysine 477 in vitro. Proteomic and immunoprecipitation analyses further reveal that the SIM3 motif is required for the EBNA1-mediated inhibitory effects on SUMO2-modified STUB1, SUMO2-mediated degradation of USP7, and SUMO1-modified KAP1. Deletion of the EBNASIM motif leads to functional loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxic stress induces the SUMO2 modification of EBNA1, and in turn the dissociation of EBNA1 with STUB1, KAP1 and USP7 to increase the SUMO1 modification of both STUB1 and KAP1 for reactivation of lytic replication. Therefore, the EBNA1SIM motif plays an essential role in EBV latency and is a potential therapeutic target against EBV-associated cancers.

Suppression of a Subset of Interferon-Induced Genes by Human Papillomavirus Type 16 E7 via a Cyclin Dependent Kinase 8-Dependent Mechanism.

Persistent infection by human papillomaviruses (HPVs), small, double-stranded DNA viruses that infect keratinocytes of the squamous epithelia, can lead to the development of cervical and other cancers. The viral oncoprotein E7 contributes to viral persistence in part by regulating host gene expression through binding host transcriptional regulators, although mechanisms responsible for E7-mediated transcriptional regulation are incompletely understood. Type I IFN signaling promotes the expression of anti-viral genes, called interferon-stimulated genes (ISGs), through the phosphorylation and activation of STAT1. In this study, we have observed that the CR3 domain of E7 contributes to the episomal maintenance of viral genomes. Transcriptome analysis revealed that E7 transcriptionally suppresses a subset of ISGs but not through regulation of STAT1 activation. Instead, we discovered that E7 associates with Mediator kinase CDK8 and this is correlated with the recruitment of CDK8 to ISG promoters and reduced ISG expression. E7 fails to suppress ISGs in the absence of CDK8, indicating that CDK8 function contributes to the suppression of ISGs by E7. Altogether, E7/CDK8 association may be a novel mechanism by which E7 inhibits innate immune signaling.

Vaccine protection against murid herpesvirus-4 is maintained when the priming virus lacks known latency genes.

γ-Herpesviruses establish latent infections of lymphocytes and drive their proliferation, causing cancers and motivating a search for vaccines. Effective vaccination against murid herpesvirus-4 (MuHV-4)-driven lymphoproliferation by latency-impaired mutant viruses suggests that lytic access to the latency reservoir is a viable target for control. However, the vaccines retained the immunogenic MuHV-4 M2 latency gene. Here, a strong reduction in challenge virus load was maintained when the challenge virus lacked the main latency-associated CD8+ T-cell epitope of M2, or when the vaccine virus lacked M2 entirely. This protection was maintained also when the vaccine virus lacked both episome maintenance and the genomic region encompassing M1, M2, M3, M4 and ORF4. Therefore, protection did not require immunity to known MuHV-4 latency genes. As the remaining vaccine virus genes have clear homologs in human γ-herpesviruses, this approach of deleting viral latency genes could alsobe applied to them, to generate safe and effective vaccines against humandisease.

Control of Viral Latency by Episome Maintenance Proteins.

The human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) share the common property of persisting as multicopy episomes in the nuclei of rapidly dividing host cells. These episomes form the molecular basis for viral latency and are etiologically linked to virus-associated cancers. Episome maintenance requires epigenetic programming to ensure the proper control of viral gene expression, DNA replication, and genome copy number. For these viruses, episome maintenance requires a dedicated virus-encoded episome maintenance protein (EMP), namely LANA (KSHV), EBNA1 (EBV), and E2 (HPV). Here, we review common features of these viral EMPs and discuss recent advances in understanding how they contribute to the epigenetic control of viral episome maintenance during latency.

Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells.

Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781–1320, 1201–1740, and 1621–2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.

Reduction of Kaposi's Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), an AIDS-defining cancer in HIV-1-infected individuals or immune-suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV's ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome but also Epstein-Barr virus (EBV) which, similar to KSHV, belongs to the Gammaherpesvirus family. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication, and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver a LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of an adenovirus vector might confer potential in vivo applications of LANA-specific Cas9 against KSHV infection and KS.IMPORTANCE The ability for Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potential benefits in developing countries where the viral cancer burden is high.