Identification of a sub-population of B cells that proliferates after infection with epstein-barr virus

<p>Abstract</p> <p>Background</p> <p>Epstein-Barr virus (EBV)-driven B cell proliferation is critical to its subsequent persistence in the host and is a key event in the development of EBV-associated B cell diseases. Thus, inquiry into early cellular events that precede EBV-driven proliferation of B cells is essential for understanding the processes that can lead to EBV-associated B cell diseases.</p> <p>Methods</p> <p>Infection with high titers of EBV of mixed, primary B cells in different stages of differentiation occurs during primary EBV infection and in the setting of T cell-immunocompromise that predisposes to development of EBV-lymphoproliferative diseases. Using an <it>ex vivo </it>system that recapitulates these conditions of infection, we correlated expression of selected B cell-surface markers and intracellular cytokines with expression of EBV latency genes and cell proliferation.</p> <p>Results</p> <p>We identified CD23, CD58, and IL6, as molecules expressed at early times after EBV-infection. EBV differentially infected B cells into two distinct sub-populations of latently infected CD23⁺cells: one fraction, marked as CD23^hiCD58⁺IL6^-by day 3, subsequently proliferated; another fraction, marked as CD23^loCD58⁺, expressed IL6, a B cell growth factor, but failed to proliferate. High levels of LMP1, a critical viral oncoprotein, were expressed in individual CD23^hiCD58⁺and CD23^loCD58⁺cells, demonstrating that reduced levels of LMP1 did not explain the lack of proliferation of CD23^loCD58⁺cells. Differentiation stage of B cells did not appear to govern this dichotomy in outcome either. Memory or naïve B cells did not exclusively give rise to either CD23^hior IL6-expressing cells; rather memory B cells gave rise to both sub-populations of cells.</p> <p>Conclusions</p> <p>B cells are differentially susceptible to EBV-mediated proliferation despite expression of viral gene products known to be critical for continuous B cell growth. Cellular events, in addition to viral gene expression, likely play a critical role in determining the outcome of EBV infection. By indentifying cells predicted to undergo EBV-mediated proliferation, our study provides new avenues of investigation into EBV pathogenesis.</p

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7

Virology under the microscope—a call for rational discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology

Inflammasome, the Constitutive Heterochromatin Machinery, and Replication of an Oncogenic Herpesvirus

The success of long-term host–virus partnerships is predicated on the ability of the host to limit the destructive potential of the virus and the virus’s skill in manipulating its host to persist undetected yet replicate efficiently when needed. By mastering such skills, herpesviruses persist silently in their hosts, though perturbations in this host–virus equilibrium can result in disease. The heterochromatin machinery that tightly regulates endogenous retroviral elements and pericentromeric repeats also silences invading genomes of alpha-, beta-, and gammaherpesviruses. That said, how these viruses disrupt this constitutive heterochromatin machinery to replicate and spread, particularly in response to disparate lytic triggers, is unclear. Here, we review how the cancer-causing gammaherpesvirus Epstein–Barr virus (EBV) uses the inflammasome as a security system to alert itself of threats to its cellular home as well as to flip the virus-encoded lytic switch, allowing it to replicate and escape in response to a variety of lytic triggers. EBV provides the first example of an infectious agent able to actively exploit the inflammasome to spark its replication. Revealing an unexpected link between the inflammasome and the epigenome, this further brings insights into how the heterochromatin machinery uses differential strategies to maintain the integrity of the cellular genome whilst guarding against invading pathogens. These recent insights into EBV biology and host–viral epigenetic regulation ultimately point to the NLRP3 inflammasome as an attractive target to thwart herpesvirus reactivation

Inflammation and Epstein–Barr Virus at the Crossroads of Multiple Sclerosis and Post-Acute Sequelae of COVID-19 Infection

Recent studies have strengthened the evidence for Epstein–Barr Virus (EBV) as an important contributing factor in the development of multiple sclerosis (MS). Chronic inflammation is a key feature of MS. EBV+ B cells can express cytokines and exosomes that promote inflammation, and EBV is known to be reactivated through the upregulation of cellular inflammasomes. Inflammation is a possible cause of the breakdown of the blood–brain barrier (BBB), which allows the infiltration of lymphocytes into the central nervous system. Once resident, EBV+ or EBV-specific B cells could both plausibly exacerbate MS plaques through continued inflammatory processes, EBV reactivation, T cell exhaustion, and/or molecular mimicry. Another virus, SARS-CoV-2, the cause of COVID-19, is known to elicit a strong inflammatory response in infected and immune cells. COVID-19 is also associated with EBV reactivation, particularly in severely ill patients. Following viral clearance, continued inflammation may be a contributor to post-acute sequelae of COVID-19 infection (PASC). Evidence of aberrant cytokine activation in patients with PASC supports this hypothesis. If unaddressed, long-term inflammation could put patients at risk for reactivation of EBV. Determining mechanisms by which viruses can cause inflammation and finding treatments for reducing that inflammation may help reduce the disease burden for patients suffering from PASC, MS, and EBV diseases

Chloroquine triggers Epstein-Barr virus replication through phosphorylation of KAP1/TRIM28 in Burkitt lymphoma cells.

Trials to reintroduce chloroquine into regions of Africa where P. falciparum has regained susceptibility to chloroquine are underway. However, there are long-standing concerns about whether chloroquine increases lytic-replication of Epstein-Barr virus (EBV), thereby contributing to the development of endemic Burkitt lymphoma. We report that chloroquine indeed drives EBV replication by linking the DNA repair machinery to chromatin remodeling-mediated transcriptional repression. Specifically, chloroquine utilizes ataxia telangiectasia mutated (ATM) to phosphorylate the universal transcriptional corepressor Krüppel-associated Box-associated protein 1/tripartite motif-containing protein 28 (KAP1/TRIM28) at serine 824 -a mechanism that typically facilitates repair of double-strand breaks in heterochromatin, to instead activate EBV. Notably, activation of ATM occurs in the absence of detectable DNA damage. These findings i) clarify chloroquine's effect on EBV replication, ii) should energize field investigations into the connection between chloroquine and endemic Burkitt lymphoma and iii) provide a unique context in which ATM modifies KAP1 to regulate persistence of a herpesvirus in humans

Chloroquine induces phosphorylation of KAP1 at S824 and activates EBV lytic cycle in Burkitt lymphoma cells.

<p><b>A.</b> HH514-16 BL cells were treated with chloroquine (CQ) or chloroquine plus KU-55933 (CQ+KU) and harvested at 24 hours followed by staining with anti-phospho KAP1 (S824) plus anti-ZEBRA antibodies and visualized at 1000X magnification. <b>B.</b> HH514-16 BL cells were treated with chloroquine and harvested at different times post-treatment for immunoblotting with antibodies as indicated. <b>C.</b> HH514-16 cells were transfected with pFLAG-CMV2-KAP1 (wt) or pFLAG-CMV2-KAP1-S824A. After 48 hours, cells were treated with chloroquine or left untreated for another 48 hours and harvested for immunoblotting with indicated as antibodies. Numbers below bands indicate relative amounts of ZEBRA after normalization to β-actin. <b>D.</b> HH514-16 cells were treated with chloroquine (black bars) or left untreated (open bars), harvested 24 hours after treatment and relative levels of transcripts from EBV lytic genes <i>BZLF1</i>, <i>BMRF1</i> and <i>BFRF3</i> were determined by qRT-PCR after normalization to 18S rRNA using the ΔΔC_T method. <b>E.</b> HH514-16 cells were untreated (open bar), treated with chloroquine (CQ; black bar: 10μM; horizontal striped bar: 200μM) or NaB (vertical striped bar), released virus particles were pelleted from supernatant 7 days later, treated with DNase, and quantified using q-PCR; ND: not detectable. <b>F.</b> BL cell lines Jijoye and Raji were treated with chloroquine (CQ) and harvested at different times post-treatment for immunoblotting with antibodies as indicated. Error bars for D and E represent 3 technical replicates from 2 experiments.</p

Chloroquine induces phosphorylation of KAP1 at S824 and activates EBV lytic cycle in lymphoblastoid cells.

<p><b>A.</b> Lymphoblastoid cells were treated with chloroquine (CQ) and harvested at different times post-treatment for immunoblotting with antibodies as indicated. <b>B.</b> Lymphoblastoid cells were untreated (open bar), treated with 10μM CQ (black bar) or 100μM CQ (horizontal striped bar) every 3 days, released virus particles were pelleted from supernatant 9 days later, treated with DNase and quantified using q-PCR. ND: not detectable; error bars represent 3 technical replicates from 2 experiments.</p