Pavel A. Nikitin

An ATM/Chk2-Mediated DNA Damage-Responsive Signaling Pathway Suppresses Epstein-Barr Virus Transformation of Primary Human B Cells

SUMMARY Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication nor did the DDR marks co-localize with latent episomes. Rather, a transient period of EBV-induced hyper-proliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoproteins EBNA3C was required to attenuate the EBV-induced DNA damage response We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR which is attenuated by viral latency products to induce cell immortalization.

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Significance Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses after congenital infection. gH/gL/gO and Pentamer are targets for neutralizing antibodies. We show that gO and UL128/UL130/UL131A bind to the same site on gH/gL through formation of a disulfide bond with gL-Cys144. The alternative use of this binding site by either gO or the ULs may provide a mechanism for cell tropism modulation. Our analysis reveals that gH/gL antigenic sites are conserved among gH/gL, gH/gL/gO, and Pentamer, whereas gH/gL/gO- and Pentamer-specific neutralizing antibody-binding sites are located in the gH/gL N terminus protrusion that contains the gO and the UL subunits. These data support the development of vaccines and antibody therapeutics against HCMV. Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.

Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody

Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection.

The DNA damage response in viral-induced cellular transformation

The DNA damage response (DDR) has emerged as a critical tumour suppressor pathway responding to cellular DNA replicative stress downstream of aberrant oncogene over-expression. Recent studies have now implicated the DDR as a sensor of oncogenic virus infection. In this review, we discuss the mechanisms by which tumour viruses activate and also suppress the host DDR. The mechanism of tumour virus induction of the DDR is intrinsically linked to the need for these viruses to promote an S-phase environment to replicate their nucleic acid during infection. However, inappropriate expression of viral oncoproteins can also activate the DDR through various mechanisms including replicative stress, direct interaction with DDR components and induction of reactive oxygen species. Given the growth-suppressive consequences of activating the DDR, tumour viruses have also evolved mechanisms to attenuate these pathways. Aberrant expression of viral oncoproteins may therefore promote tumourigenesis through increased somatic mutation and aneuploidy due to DDR inactivation. This review will focus on the interplay between oncogenic viruses and the DDR with respect to cellular checkpoint control and transformation.

Metabolic stress is a barrier to Epstein–Barr virus-mediated B-cell immortalization

Significance Epstein–Barr virus (EBV) was the first human tumor virus discovered. Although nearly all adults are infected with EBV, very few go on to develop disease, for reasons that we are only beginning to understand. Infection with EBV induces a period of very rapid cell division, which requires an increased supply of metabolites, such as nucleotides, amino acids, and lipids. We found that EBV-infected cells that are unable to meet this increased metabolic demand are forced to stop proliferating and undergo a permanent growth arrest called senescence. Epstein–Barr virus (EBV) is an oncogenic herpesvirus that has been causally linked to the development of B-cell and epithelial malignancies. Early after infection, EBV induces a transient period of hyperproliferation that is suppressed by the activation of the DNA damage response and a G1/S-phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation and integrated gene expression and metabolic profiling to gain a better understanding of the pathways that attenuate immortalization. We found that the arrested cells have a reduced level of mitochondrial respiration and a decrease in the expression of genes involved in the TCA cycle and oxidative phosphorylation. Indeed, the growth arrest in early infected cells could be rescued by supplementing the TCA cycle. Arrested cells were characterized by an increase in the expression of p53 pathway gene targets, including sestrins leading to activation of AMPK, a reduction in mTOR signaling, and, consequently, elevated autophagy that was important for cell survival. Autophagy was also critical to maintain early hyperproliferation during metabolic stress. Finally, in assessing the metabolic changes from early infection to long-term outgrowth, we found concomitant increases in glucose import and surface glucose transporter 1 (GLUT1) levels, leading to elevated glycolysis, oxidative phosphorylation, and suppression of basal autophagy. Our study demonstrates that oncogene-induced senescence triggered by a combination of metabolic and genotoxic stress acts as an intrinsic barrier to EBV-mediated transformation.

Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest.

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection

Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis. DOI: http://dx.doi.org/10.7554/eLife.22509.001

In Vitro Characterization of Human Cytomegalovirus-Targeting Therapeutic Monoclonal Antibodies LJP538 and LJP539

ABSTRACT Human cytomegalovirus (HCMV) infection is usually benign in healthy individuals but can cause life-threatening disease in those with compromised immune systems. Approved drugs available to treat HCMV disease, including ganciclovir, cidofovir, and foscarnet, have significant toxicities that limit their use in certain patient populations. LJP538 and LJP539 are human monoclonal antibodies that are being evaluated as immunoglobulin therapeutics. The antibodies target glycoproteins gB and the gH/gL/UL128/UL130/UL131a pentameric complex, respectively. Here we present an in vitro characterization of these antibodies. We show that LJP538 and LJP539 are more potent than a marketed immunoglobulin at inhibiting HCMV infection of various cell lines relevant to pathogenesis. We find that LJP538 and LJP539 are active against a panel of clinical isolates in vitro and demonstrate minor-to-moderate synergy in combination. Passage of HCMV in the presence of LJP538 or LJP539 alone resulted in resistance-associated mutations that mapped to the target genes. However, no loss of susceptibility to the combination of antibodies was observed for >400 days in culture. Finally, the binding regions of LJP538 and LJP539 are conserved among clinical isolates. Taken together, these data support the use of LJP538 and LJP539 in combination for clinical trials in HCMV patients.

Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. ABSTRACT Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

Epstein-Barr virus induces adhesion molecule CD226 (DNAM-1) expression during primary B cell transformation into lymphoblastoid cell lines

Epstein-Barr virus (EBV), an oncogenic herpesvirus associated with Burkitt’s lymphoma and other AIDS-related B cell malignancies, transforms primary human B cells into lymphoblastoid cell lines (LCLs) ex vivo. As LCLs express viral gene products similar to those found in EBV-mediated cancers, LCLs provide a practical model for tumorigenesis. Previous unpublished findings from our lab indicate that LCLs constitutively express the adhesion molecule CD226 (DNAM-1), found on virtually all peripheral blood NK cells, T cells, and monocytes, but only a small subset (~3%) of B cells. Although CD226 is known to mediate T-cell differentiation and cytotoxicity, NK cell cytotoxicity, NKT cell apoptosis, and monocyte extravasion, CD226 function in B cells remains relatively unstudied. Biochemically, CD226 functions to support the interaction between the intracellular adhesion molecules LFA-1 and ICAM-1. Here, we demonstrate that EBV specifically induces CD226 expression in primary human B cells and EBV-negative B lymphoblasts during viral-mediated proliferation and outgrowth. EBV infection of primary B cells increased CD226 surface expression 5-fold during early proliferation and approximately 30-fold upon transformation into LCLs. EBV-converted Burkitt’s lymphoma cells constitutively express CD226, while EBV-negative B cell lymphomas do not. Additionally, we demonstrate that LMP-1, an EBV latency III membrane oncoprotein, induces CD226 expression in EBV-negative Burkitt’s lymphoma cells. Finally, we demonstrate that the NFκB pathway regulates CD226 expression. Indeed, B cell lymphomas with high NFκB activity (activated B cell-like diffuse large B-cell lymphomas) express CD226 at higher levels than B cell lymphomas with low NFκB activity (germinal center B cell-like diffuse large B cell lymphomas). As CD226 supports the interaction between LFA-1 and ICAM-1, which is critical to maintain the constitutive aggregation of EBV-transformed B cells, we propose that EBV-mediated induction of CD226 drives cell-cell contact ensuring B cell survival. These data suggest that CD226, a newly identified EBV-induced cell adhesion molecule, may play a key role in the pathogenesis of AIDS-associated and other B cell lymphomas.