Episomal Maintenance Research Articles

Minichromosome Maintenance Proteins Cooperate with LANA during the G1/S Phase of the Cell Cycle To Support Viral DNA Replication.

Latency-associated nuclear antigen (LANA) is essential for maintaining the viral genome by regulating replication and segregation of the viral episomes. The virus maintains 50 to 100 episomal copies during latency and replicates in synchrony with the cellular DNA of the infected cells. Since virus lacks its own replication machinery, it utilizes the cellular proteins for replication and maintenance, and LANA has been shown to make many of these proteins available for replication by directly recruiting them to the viral origin of replication within the terminal repeat (TR) region. Our studies identified members of the minichromosome maintenance (MCM) complex as potential LANA-interacting proteins. Here, we show that LANA specifically interacts with the components of the MCM complex, primarily during the G1/S phase of the cell cycle. MCM3 and -4 of the MCM complex specifically bound to the amino-terminal domain, while MCM6 bound to both the amino- and carboxyl-terminal domains of LANA. The MCM binding region in the N-terminal domain mapped to the chromatin binding domain (CBD). LANA with point mutations in the carboxyl-terminal domain identified an MCM6 binding domain, and overexpression of that domain (amino acids [aa] 1100 to 1150) abolished TR replication. Introduction of a peptide encompassing the LANA aa 1104 to 1123 reduced MCM6 association with LANA and TR replication. Moreover, a recombinant Kaposi's sarcoma-associated herpesvirus (KSHV) expressing LANA with a deletion of aa 1100 to 1150 (BAC16Δ1100-1150, where BAC is bacmid) showed reduced replication and persistence of viral genome copies compared to levels with the wild-type BAC16. Additionally, the role of MCMs in viral replication was confirmed by depleting MCMs and assaying transient and long-term maintenance of the viral episomes. The recruitment of MCMs to the replication origins through LANA was demonstrated through chromatin immunoprecipitation and isolation of proteins on nascent replicated DNA (iPOND). These data clearly show the role of MCMs in latent DNA replication and the potential for targeting the C-terminal domain of LANA to block viral persistence.IMPORTANCE LANA-mediated latent DNA replication is essential for efficient maintenance of KSHV episomes in the host. During latency, virus relies on the host cellular machinery for replication, which occurs in synchrony with the cellular DNA. LANA interacts with the components of multiple cellular pathways, including cellular replication machinery, and recruits them to the viral origin for DNA replication. In this study, we characterize the interactions between LANA and minichromosome maintenance (MCM) proteins, members of the cellular replication complex. We demonstrated a cell cycle-dependent interaction between LANA and MCMs and determined their importance for viral genome replication and maintenance through biochemical assays. In addition, we mapped a 50-amino acid region in LANA which was capable of abrogating the association of MCM6 with LANA and blocking DNA replication. We also detected LANA along with MCMs at the replication forks using a novel approach, isolation of proteins on nascent DNA (iPOND).

Kaposi's Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA's ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence.IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.

Defining early events of Epstein-Barr virus (EBV) infection in immortalized nasopharyngeal epithelial cells using cell-free EBV infection.

Epstein-Barr virus (EBV) infection is strongly associated with nasopharyngeal carcinoma, a common cancer in Southeast Asia and certain regions of Africa. However, the dynamics of EBV episome maintenance in infected nasopharyngeal epithelial (NPE) cells remain largely undefined. Here, we report the establishment of a highly efficient cell-free EBV infection method for NPE cells. By using this method, we have defined some of the dynamic events involved in the early stage of EBV infection in NPE cells. We report, for the first time, a rapid loss of EBV copies from infected NPE cells during the first 12-72 h post-infection. The rate of EBV loss slowed at later stages of infection. Live cell imaging revealed that the freshly infected NPE cells were delayed in entry into mitosis compared with uninfected cells. Freshly infected NPE cells transcribed significantly higher levels of lytic EBV genes BZLF1 and BMRF1 yet significantly lower levels of EBER1/2 than stably infected NPE cells. Notably, there were very low or undetectable levels of protein expressions of EBNA1, LMP1, Zta and Rta in freshly infected NPE cells, whereas EBNA1 and LMP1 proteins were readily detected in stable EBV-infected NPE cells. The kinetics of EBV loss and the differential EBV gene expression profiles between freshly and stably infected NPE cells are in line with the suggestion of epigenetic changes in the EBV genome that affect viral gene expression and the adaptation of host cells to EBV infection to maintain persistent EBV infection in NPE cells.

LANA oligomeric architecture is essential for KSHV nuclear body formation and viral genome maintenance during latency.

The molecular basis for the formation of functional, higher-ordered macro-molecular domains is not completely known. The Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) genome forms a super-molecular domain structure during latent infection that is strictly dependent on the DNA binding of the viral nuclear antigen LANA to the viral terminal repeats (TR). LANA is known to form oligomeric structures that have been implicated in viral episome maintenance. In this study, we show that the LANA oligomerization interface is required for the formation of higher-order nuclear bodies that partially colocalize with DAXX, EZH2, H3K27me3, and ORC2 but not with PML. These nuclear bodies assemble at the periphery of condensed cellular chromosomes during mitotic cell division. We demonstrate that the LANA oligomerization interface contributes to the cooperative DNA binding at the viral TR and the recruitment of ORC to the viral episome. Oligomerization mutants failed to auto-regulate LANA/ORF73 transcription, and this correlated with the loss of a chromosome conformational DNA-loop between the TR and LANA promoter. Viral genomes with LANA oligomerization mutants were subject to genome rearrangements including the loss of subgenomic DNA. Our data suggests that LANA oligomerization drives stable binding to the TR and formation of an epigenetically stable chromatin architecture resulting in higher-order LANA nuclear bodies important for viral genome integrity and long-term episome persistence.

Epstein-Barr Virus Genome Replication as a Molecular Target for Cancer Therapy

Epstein-Barr virus (EBV), a human oncogenic virus, is a B cell-tropic herpesvirus and has the ability to immortalize normal B cells during latent infection. The Epstein-Barr nuclear antigen 1 (EBNA1) protein of EBV is expressed in the most EBV latently infected cells and binds to a specific viral genome region termed "oriP" (origin of plasmid replication) to maintain the stability of the approximately 170 kb double-stranded circular virus genomic DNA (episome) in cells. EBV elimination is thought to inhibit progression of EBV-associated malignancies, and the EBNA1-dependent mechanisms for EBV episome replication and maintenance are considered to be novel molecular targets for anti-EBV therapy. We have explored small-molecule compounds that can inhibit the binding between EBNA1 protein and oriP and found one pyrrole imidazole polyamide named DSE3 which can also inhibit EBV-mediated immortalization of normal B cells. These data suggested that an EBNA1-targeting strategy could be useful to combat EBV-associated malignancies.

The dynamics of integration, viral suppression and cell-cell transmission in the development of occult Hepatitis B virus infection

BackgroundOut of several phases of HBV infection, the least understood phase is occult hepatitis B virus infection. The paucity of data due to non-availability of biological tissues and the prerequisite of ultra-sensitive assays for the detection of occult hepatitis B virus infection prompted us to utilize mathematical modeling in determining mechanisms that lead to occult hepatitis B virus infection and characteristics of HBV infection during occult hepatitis B virus infection. MethodsWe proposed two mathematical models (M1 and M2), considering two different phenomenon for episomal maintenance and accumulation of covalently closed circular DNA (cccDNA) in infected hepatocytes: (i) M1 – recirculation of the relaxed circular DNA/double-stranded linear DNA from cytoplasm to the nucleus, and (ii) M2 – reinfection of infected hepatocytes with virions. We further incorporated the dynamics of integrated Hepatitis B virus DNA (iHBV) to investigate its role in the development of occult hepatitis B virus infection. ResultsThe analysis showed that the main mechanism for the spread of infection during occult hepatitis B virus infection is cell-to-cell transmission and not cell-free virus transmission. A significant viral suppression (of at least 99% from its peak production values) was essential but not sufficient in the development of occult hepatitis B virus infection under M1; however under M2, the viral suppression was neither sufficient nor essential as the inhibition of the production of HBsAg without viral suppression can also explain the development of occult hepatitis B virus infection. Our analysis also revealed that occult hepatitis B virus infection seropositive cases are more likely to progress into liver cirrhosis compared to occult hepatitis B virus infection seronegative cases. The iHBV was found to be mostly silent (by either being absent or non-productive for HBsAg) during occult hepatitis B virus infection. ConclusionThe viral suppression is neither essential nor sufficient to explain the development of occult hepatitis B virus infection on its own. Not only the viral suppression but the inhibition -of the production and the export of HBsAg from cccDNA and iHBV also plays an important role in the development of occult hepatitis B virus infection. This is the first study, which incorporates the dynamics of iHBV and shows that HBV primarily spreads via cell-cell transmission during occult hepatitis B virus infection.

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.

A new cell culture model to genetically dissect the complete human papillomavirus life cycle.

Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

Cross-species conservation of episome maintenance provides a basis for in vivo investigation of Kaposi's sarcoma herpesvirus LANA.

Many pathogens, including Kaposi’s sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens.

Structural and Functional Basis for an EBNA1 Hexameric Ring in Epstein-Barr Virus Episome Maintenance.

Epstein-Barr virus (EBV) establishes a stable latent infection that can persist for the life of the host. EBNA1 is required for the replication, maintenance, and segregation of the latent episome, but the structural features of EBNA1 that confer each of these functions are not completely understood. Here, we have solved the X-ray crystal structure of an EBNA1 DNA-binding domain (DBD) and discovered a novel hexameric ring oligomeric form. The oligomeric interface pivoted around residue T585 as a joint that links and stabilizes higher-order EBNA1 complexes. Substitution mutations around the interface destabilized higher-order complex formation and altered the cooperative DNA-binding properties of EBNA1. Mutations had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance with OriP. We found that one naturally occurring polymorphism in the oligomer interface (T585P) had greater cooperative DNA binding in vitro, minor defects in DNA replication, and pronounced defects in episome maintenance. The T585P mutant was compromised for binding to OriP in vivo as well as for assembling the origin recognition complex subunit 2 (ORC2) and trimethylated histone 3 lysine 4 (H3K4me3) at OriP. The T585P mutant was also compromised for forming stable subnuclear foci in living cells. These findings reveal a novel oligomeric structure of EBNA1 with an interface subject to naturally occurring polymorphisms that modulate EBNA1 functional properties. We propose that EBNA1 dimers can assemble into higher-order oligomeric structures important for diverse functions of EBNA1.IMPORTANCE Epstein-Barr virus is a human gammaherpesvirus that is causally associated with various cancers. Carcinogenic properties are linked to the ability of the virus to persist in the latent form for the lifetime of the host. EBNA1 is a sequence-specific DNA-binding protein that is consistently expressed in EBV tumors and is the only viral protein required to maintain the viral episome during latency. The structural and biochemical mechanisms by which EBNA1 allows the long-term persistence of the EBV genome are currently unclear. Here, we have solved the crystal structure of an EBNA1 hexameric ring and characterized key residues in the interface required for higher-order complex formation and long-term plasmid maintenance.

S/MAR Element Facilitates Episomal Long-Term Persistence of Adeno-Associated Virus Vector Genomes in Proliferating Cells.

Adeno-associated virus (AAV) vectors are one of the most frequently applied gene transfer systems in research and human clinical trials. Since AAV vectors do not possess an integrase activity, application is restricted to terminally differentiated tissues if transgene expression is required long term. To overcome this limitation and to generate AAV vectors that persist episomally in dividing cells, AAV vector genomes were equipped with a scaffold/matrix attachment region (S/MAR). After a mild antibiotic selection, cells transduced with AAV-S/MAR established colonies that maintained long-term transgene expression (>50 population doublings) from replicating AAV vector episomes in the absence of further selection. Unexpectedly, with a lesser but still significant efficiency, the control vector (AAV-ΔS/MAR), a standard single-stranded AAV vector, also established stable transgene-expressing colonies, most of which were maintained as replicating episomes rather than integrated vector genomes. Thus, based on the result in HeLa cells, it is concluded that AAV vector genomes per se possess the ability to establish episomal maintenance in proliferating cells, a feature that can be enhanced by incorporation of a foreign genomic element such as an S/MAR element.

FANCD2 Binds Human Papillomavirus Genomes and Associates with a Distinct Set of DNA Repair Proteins to Regulate Viral Replication.

ABSTRACTThe life cycle of human papillomavirus (HPV) is dependent on the differentiation state of its host cell. HPV genomes are maintained as low-copy episomes in basal epithelial cells and amplified to thousands of copies per cell in differentiated layers. Replication of high-risk HPVs requires the activation of the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) DNA repair pathways. The Fanconi anemia (FA) pathway is a part of the DNA damage response and mediates cross talk between the ATM and ATR pathways. Our studies show that HPV activates the FA pathway, leading to the accumulation of a key regulatory protein, FANCD2, in large nuclear foci. These HPV-dependent foci colocalize with a distinct population of DNA repair proteins, including ATM components γH2AX and BRCA1, but infrequently with p-SMC1, which is required for viral genome amplification in differentiated cells. Furthermore, FANCD2 is found at viral replication foci, where it is preferentially recruited to viral genomes compared to cellular chromosomes and is required for maintenance of HPV episomes in undifferentiated cells. These findings identify FANCD2 as an important regulator of HPV replication and provide insight into the role of the DNA damage response in the differentiation-dependent life cycle of HPV.

Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr Virus episomal latency.

Epstein-Barr Virus (EBV) latent infection is a causative co-factor for endemic Nasopharyngeal Carcinoma (NPC). NPC-associated variants have been identified in EBV-encoded nuclear antigen EBNA1. Here, we solve the X-ray crystal structure of an NPC-derived EBNA1 DNA binding domain (DBD) and show that variant amino acids are found on the surface away from the DNA binding interface. We show that NPC-derived EBNA1 is compromised for DNA replication and episome maintenance functions. Recombinant virus containing the NPC EBNA1 DBD are impaired in their ability to immortalize primary B-lymphocytes and suppress lytic transcription during early stages of B-cell infection. We identify Survivin as a host protein deficiently bound by the NPC variant of EBNA1 and show that Survivin depletion compromises EBV episome maintenance in multiple cell types. We propose that endemic variants of EBNA1 play a significant role in EBV-driven carcinogenesis by altering key regulatory interactions that destabilize latent infection.

The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment.

ABSTRACTIn papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. IMPORTANCE Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection is a risk factor for cancer development and is partly achieved by the attachment of viral DNA to cellular chromatin during cell division. The HPV E2 protein plays a critical role in this tethering by binding simultaneously to the viral genome and to chromatin during mitosis. We previously showed that the cellular DNA helicase ChlR1 is required for loading of the bovine papillomavirus E2 protein onto chromatin during DNA synthesis. Here we identify a mutation in HPV16 E2 that abrogates interaction with ChlR1, and we show that ChlR1 regulates the chromatin association of HPV16 E2 and that this virus-host interaction is essential for viral episome maintenance.

Kaposi's Sarcoma Herpesvirus Genome Persistence.

Kaposi’s sarcoma-associated herpesvirus (KSHV) has an etiologic role in Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA). LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA’s role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease.

Refinement of the Diatom Episome Maintenance Sequence and Improvement of Conjugation-Based DNA Delivery Methods.

Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here, we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in Escherichia coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in Phaeodactylum tricornutum. While various combinations of these individual elements enable efficient conjugation and high exconjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3′ end of HIS3 produced the highest number of diatom exconjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a high-throughput option utilizing 12-well plates and plating methods that improve exconjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance and demonstrate options for flexible vector design.

HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus.

ABSTRACTEpstein-Barr virus (EBV) establishes latent infections as multicopy episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependent C promoter (Cp) and Q promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV episomes from Burkitt's lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation.IMPORTANCE EBV latency is associated with several human cancers. Viral latent cycle gene expression is regulated by the epigenetic control of the OriP enhancer region. Here, we show that cellular factors OCT2 and HCF1 bind OriP in association with EBNA1 to maintain elevated histone H3K4me3 and transcriptional enhancer function. HCF1 is known as a transcriptional coactivator of herpes simplex virus (HSV) immediate early (IE) transcription, suggesting that OriP enhancer shares aspects of HSV IE transcription control.

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal genetransfer.

Chimeric antigen receptor (CAR) T‐cell therapy is a new successful treatment for refractory B‐cell leukemia. Successful therapeutic outcome depends on long‐term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long‐term cell engineering method using non‐integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV‐S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV‐S/MAR‐engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19+ target cell recognition as LV‐engineered T cells and are as effective in controlling tumor growth in vivo. We propose that NILV‐S/MAR vectors are superior to current options as they enable long‐term transgene expression without the risk of insertional mutagenesis and genotoxicity.

Ribosome Protein L4 is essential for Epstein–Barr Virus Nuclear Antigen 1 function

Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection.

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases.