Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection.

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV's lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV's lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV's lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

Epstein-Barr virus reprograms human B lymphocytes immediately in the prelatent phase of infection.

Epstein-Barr virus (EBV) is a human tumor virus and a model of herpesviral latency. The virus efficiently infects resting human B lymphocytes and induces their continuous proliferation in vitro, which mimics certain aspects of EBV's oncogenic potential in vivo. How lymphoblastoid cell lines (LCLs) evolve from the infected lymphocytes is uncertain. We conducted a systematic time-resolved longitudinal study of cellular functions and transcriptional profiles of newly infected naïve primary B lymphocytes. EBV reprograms the cells comprehensively and globally. Rapid and extensive transcriptional changes occur within 24 h and precede any metabolic and phenotypic changes. Within 72 h, the virus activates the cells, changes their phenotypes with respect to cell size, RNA, and protein content, and induces metabolic pathways to cope with the increased demand for energy, supporting an efficient cell cycle entry on day 3 postinfection. The transcriptional program that EBV initiates consists of 3 waves of clearly discernable clusters of cellular genes that peak on day 2, 3, or 4 and regulate RNA synthesis, metabolic pathways, and cell division, respectively. Upon onset of cell doublings on day 4, the cellular transcriptome appears to be completely reprogrammed to support the proliferating cells, but 3 additional clusters of EBV-regulated genes fine-tune cell signaling, migration, and immune response pathways, eventually. Our study reveals that more than 11,000 genes are regulated upon EBV infection as naïve B cells exit quiescence to enter a germinal center-like differentiation program, which culminates in immortalized, proliferating cells that partially resemble plasmablasts and early plasma cells.

Cutting edge: miR-223 and EBV miR-BART15 regulate the NLRP3 inflammasome and IL-1ß production.

Although microRNA (miRNA) regulation of TLR signaling is well established, this has not yet been observed for NLR proteins or the inflammasomes they form. We have now validated a highly conserved miR-223 target site in the NLRP3 3'-untranslated region. miR-223 expression decreases as monocytes differentiate into macrophages, whereas NLRP3 protein increases during this time. However, overexpression of miR-223 prevents accumulation of NLRP3 protein and inhibits IL-1ß production from the inflammasome. Virus inhibition of the inflammasome is an emerging theme, and we have also identified an EBV miRNA that can target the miR-223 binding site in the NLRP3 3'-untranslated region. Furthermore, this virus miRNA can be secreted from infected B cells via exosomes to inhibit the NLRP3 inflammasome in noninfected cells. Therefore, we have identified both the first endogenous miRNA that limits NLRP3 inflammatory capacity during myeloid cell development and also a viral miRNA that takes advantage of this, limiting inflammation for its own purposes.

AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions dependent on the epigenetic state of the viral genome.

EBV, a member of the herpes virus family, is a paradigm for human tumor viruses and a model of viral latency amenable for study in vitro. It induces resting human B lymphocytes to proliferate indefinitely in vitro and initially establishes a strictly latent infection in these cells. BZLF1, related to the cellular activating protein 1 (AP-1) family of transcription factors, is the viral master gene essential and sufficient to mediate the switch to induce the EBV lytic phase in latently infected B cells. Enigmatically, after infection BZLF1 is expressed very early in the majority of primary B cells, but its early expression fails to induce the EBV lytic phase. We show that the early expression of BZLF1 has a critical role in driving the proliferation of quiescent naïve and memory B cells but not of activated germinal center B cells. BZLF1's initial failure to induce the EBV lytic phase relies on the viral DNA at first being unmethylated. We have found that the eventual and inevitable methylation of viral DNA is a prerequisite for productive infection in stably, latently infected B cells which then yield progeny virus lacking cytosine-phosphatidyl-guanosine (CpG) methylation. This progeny virus then can repeat EBV's epigenetically regulated, biphasic life cycle. Our data indicate that the viral BZLF1 protein is crucial both to establish latency and to escape from it. Our data also indicate that EBV has evolved to appropriate its host's mode of methylating DNA for its own epigenetic regulation.

Assessing the Efficacy of VLP-Based Vaccine against Epstein-Barr Virus Using a Rabbit Model.

Epstein-Barr virus (EBV) is etiologically associated with a number of malignant and non-malignant conditions. Thus, a prophylactic vaccine against this virus could help to reduce the burden of many EBV-associated diseases. Previously, we reported that an EBV virus-like particle (VLP) vaccine was highly immunogenic and produced a strong humoral response in mice. However, since EBV does not infect mice, the efficacy of the VLP in preventing EBV infection could not be addressed. Here we examined, for the first time, the efficacy of the EBV-VLP vaccine using a novel rabbit model of EBV infection. Animals vaccinated with two doses of VLP elicited higher antibody responses to total EBV antigens compared to animals receiving one dose. Vaccinated animals also elicited both IgM and IgG to EBV-specific antigens, VCA and EBNA1. Analysis of peripheral blood and spleen for EBV copy number indicated that the viral load in both of these compartments was lower in animals receiving a 2-dose vaccine. However, the VLP vaccine was ineffective in preventing EBV infection. With several other EBV vaccine candidates currently at various stages of development and testing, we believe that the rabbit model of EBV infection could be a great platform for evaluating potential candidates.

SARS-CoV-2 and Epstein-Barr Virus-like Particles Associate and Fuse with Extracellular Vesicles in Virus Neutralization Tests.

The successful development of effective viral vaccines depends on well-known correlates of protection, high immunogenicity, acceptable safety criteria, low reactogenicity, and well-designed immune monitoring and serology. Virus-neutralizing antibodies are often a good correlate of protective immunity, and their serum concentration is a key parameter during the pre-clinical and clinical testing of vaccine candidates. Viruses are inherently infectious and potentially harmful, but we and others developed replication-defective SARS-CoV-2 virus-like-particles (VLPs) as surrogates for infection to quantitate neutralizing antibodies with appropriate target cells using a split enzyme-based approach. Here, we show that SARS-CoV-2 and Epstein-Barr virus (EBV)-derived VLPs associate and fuse with extracellular vesicles in a highly specific manner, mediated by the respective viral fusion proteins and their corresponding host receptors. We highlight the capacity of virus-neutralizing antibodies to interfere with this interaction and demonstrate a potent application using this technology. To overcome the common limitations of most virus neutralization tests, we developed a quick in vitro diagnostic assay based on the fusion of SARS-CoV-2 VLPs with susceptible vesicles to quantitate neutralizing antibodies without the need for infectious viruses or living cells. We validated this method by testing a set of COVID-19 patient serum samples, correlated the results with those of a conventional test, and found good sensitivity and specificity. Furthermore, we demonstrate that this serological assay can be adapted to a human herpesvirus, EBV, and possibly other enveloped viruses.

Different roles of the human Orc6 protein in the replication initiation process.

In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1-Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test.

Neutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests currently in use are mostly surrogate tests based on direct or competitive enzyme immunoassays or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like-particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome and thus are non-infectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) showed excellent specificity and sensitivity and correlated very well with a cVNT using fully infectious SARS-CoV-2. The results also demonstrated the reduced neutralizing capacity of COVID-19 vaccinee sera against variants of concern of SARS-CoV-2 including omicron B.1.1.529, BA.1.

Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins.

In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G(1) and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.

Multiple Viral microRNAs Regulate Interferon Release and Signaling Early during Infection with Epstein-Barr Virus.

Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.IMPORTANCE Acute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors that induce the transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/ß receptor, the subsequent activation of JAK/STAT signaling pathways, and, finally, the transcription of many IFN-stimulated genes (ISGs). In sum, these cellular functions establish a so-called antiviral state in infected and neighboring cells. To counteract these cellular defense mechanisms, viruses have evolved diverse strategies and encode gene products that target antiviral responses. Among such immune-evasive factors are viral microRNAs (miRNAs) that can interfere with host gene expression. We discovered that multiple miRNAs of Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the antiviral states of immune cells, specifically B cells and plasmacytoid dendritic cells (pDCs). We identified the viral DNA genome as the activator of IFN-α and question the role of abundant EBV EBERs, that, contrary to previous reports, do not have an apparent inducing function in the IFN-I pathway early after infection.

Conditional gene vectors regulated in cis.

Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time.

Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL.

BACKGROUND: Clinically relevant methods are not available that prioritize and validate potential therapeutic targets for individual tumors, from the vast amount of tumor descriptive expression data. METHODS: We established inducible transgene expression in clinically relevant patient-derived xenograft (PDX) models in vivo to fill this gap. RESULTS: With this technique at hand, we analyzed the role of the transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive preclinical in vivo trials, we found that re-expression of wild type KLF4 reduced the leukemia load in PDX models of B-ALL, with the strongest effects being observed after conventional chemotherapy in minimal residual disease (MRD). A nonfunctional KLF4 mutant had no effect on this model. The re-expression of KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. It is of major translational relevance that azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced azacitidine-induced cell death, suggesting that azacitidine can regulate KLF4 re-expression. These results support the application of azacitidine in patients with B-ALL as a therapeutic option to regulate KLF4. CONCLUSION: Genetic engineering of PDX models allows the examination of the function of dysregulated genes like KLF4 in a highly clinically relevant translational context, and it also enables the selection of therapeutic targets in individual tumors and links their functions to clinically available drugs, which will facilitate personalized treatment in the future.

First Days in the Life of Naive Human B Lymphocytes Infected with Epstein-Barr Virus.

Epstein-Barr virus (EBV) infects and activates resting human B lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines, many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive human B lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the prelatent phase. Even EBNA1, which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a decisive parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B lymphocytes from energetically quiescent to activated cells.IMPORTANCE The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 days later, the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains, we investigated the individual contributions of EBV's multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV's prelatent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming, viral latent genes other than EBNA2 are dispensable, but some, EBNA-LP, for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

Transcriptional activation by EBV nuclear antigen 1 is essential for the expression of EBV's transforming genes.

EBV is a paradigm for human tumor viruses because, although it infects most people benignly, it also can cause a variety of cancers. Both in vivo and in vitro, EBV infects B lymphocytes in G0, induces them to become blasts, and can maintain their proliferation in cell culture or in vivo as tumors. How EBV succeeds in these contrasting cellular environments in expressing its genes that control the host has not been explained. We have genetically dissected the EBV nuclear antigen 1 (EBNA1) gene that is required for replication of the viral genome, to elucidate its possible role in the transcription of viral genes. Strikingly, EBNA1 is essential to drive transcription of EBV's transforming genes after infection of primary B lymphocytes.

Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus.

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.

Epstein-Barr virus nuclear antigen 1 evades direct immune recognition by CD4+ T helper cells.

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein regularly expressed in EBV-associated malignancies. Immune recognition of EBNA1 by CD8+ T cells is prevented by an internal glycine-alanine repeat (GAr) which blocks proteasomal degradation. To test whether EBV-infected cells could be recognized by T helper cells, human CD4+ T cell clones specific for EBNA1 were isolated from latently EBV-infected individuals. These T cells, however, failed to recognize EBV-positive target cells. To investigate whether endogenous presentation of EBNA1 epitopes on MHC class II was prevented by the GAr domain, a mutant EBV strain with an EBNA1 lacking the GAr (EBNA1DeltaGA) was generated and used to establish an Epstein-Barr virus-immortalized lymphoblastoid B cell line (LCL). The EBNA1DeltaGA LCL were not recognized by the EBNA1-specific T cell clones either, indicating that the GAr domain does not mediate this effect. Immune recognition could be restored by overexpression of EBNA1, for which at least 60-fold higher levels of both EBNA1 or EBNA1DeltaGAr protein were required. These results demonstrate that EBNA1 evades direct recognition by CD4+ T helper cells, since its steady state level is below the threshold required for efficient presentation on MHC class II. These findings have important implications for the design of immunotherapeutic approaches to target EBV-positive malignancies.