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The recently identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. How this novel beta-coronavirus virus, and coronaviruses more generally, alter cellular metabolism to support massive production of ~30 kB viral genomes and subgenomic viral RNAs remains largely unknown. To gain insights, transcriptional and metabolomic analyses are performed 8 hours after SARS-CoV-2 infection, an early timepoint where the viral lifecycle is completed but prior to overt effects on host cell growth or survival. Here, we show that SARS-CoV-2 remodels host folate and one-carbon metabolism at the post-transcriptional level to support de novo purine synthesis, bypassing viral shutoff of host translation. Intracellular glucose and folate are depleted in SARS-CoV-2-infected cells, and viral replication is exquisitely sensitive to inhibitors of folate and one-carbon metabolism, notably methotrexate. Host metabolism targeted therapy could add to the armamentarium against future coronavirus outbreaks. Viruses rely on host metabolism for replication. Here, the authors perform transcriptional and metabolomic analyses at 8 hours after SARS-CoV-2 infection and find that the virus alters host folate and one-carbon metabolism at a post-transcriptional level.

[This corrects the article DOI: 10.1371/journal.ppat.1010254.].

Funding: We acknowledge support by NIH RO1 AI137337, AI164709 and CA228700 and a Burroughs Wellcome Career Award in Medical Sciences to BEG and by NIH T32AI007245 to E.M.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. https://grants.nih.gov/grants/funding/r01.htm https://researchtraining.nih.gov/programs/training-grants/t32 https://www.bwfund.org/funding-opportunities/biomedical-sciences/career-awards-for-medical-scientists/. EBNA, Epstein–Barr nuclear antigen; EBV, Epstein–Barr virus; LCL, lymphoblastoid cell line; LMP, latent membrane protein; ncRNA, noncoding RNA; OXPHOS, oxidative phosphorylation. https://doi.org/10.1371/journal.ppat.1010254.g001 Latent EBV induction of aerobic glycolysis Within 4 days of infection, EBV highly induces glucose uptake and expression of all glycolysis enzymes at the mRNA and protein levels, in particular the first and rate-limiting enzyme hexokinase 2 [2,4,9]. EBV-driven purine and pyrimidine nucleotide metabolism Nucleotide synthesis rates are low in resting B lymphocytes, which reside in a Go cell cycle state. [...]EBV must rapidly induce purine and pyrimidine biosynthesis for genome replication as well as ribosomal RNAs to support elevated translation rates in newly infected cells [2]. ADA levels progressively increase in EBV-infected cells, particularly as they convert to the lymphoblastoid phase [27]. [...]EBV-induced ADA may serve to protect infected B cells as they transition through germinal center reactions and again in lymphoblastoid B cells, where ADA knockdown impairs proliferation in vitro [27].

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.

The Epstein-Barr virus (EBV) oncoprotein Latent Membrane Protein 1 (LMP1) is expressed in multiple malignancies and is critical for B-cell immortalization. LMP1 constitutively activates NF-κB signaling pathways, which are essential for EBV-mediated B cell transformation and for transformed B cell survival. Reverse genetic analysis revealed two LMP1 regions critical for primary human B cell immortalization, termed transformation effector site (TES) 1 and 2, which activate multiple host growth and survival pathways, in particular NF-κB. Of these, only TES1 signaling is required for B-cell transformation within the first several weeks of infection. TES1 signaling is also critical for EBV-transformed lymphoblastoid B-cell survival. However, precisely how TES1 initiates NF-κB signaling has remained incompletely understood. Here, we provide multiple lines of evidence that TES1 associates with cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2) in a tumor necrosis factor associated factor 3 (TRAF3) dependent manner. TES1 signaling drives cIAP1 autoubiquitination and targets TRAF2, cIAP1 and 2 for proteasomal degradation in a TRAF3 dependent manner. Overexpression of either cIAP1 or 2 impaired LMP1 TES1-mediated non-canonical NF-κB activation. Collectively, these studies suggest that LMP1 TES1 initiates non-canonical NF-κB signaling distinctly from CD40 and other host immunoreceptors, thereby highlighting a therapeutic target.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.

Epstein-Barr virus (EBV) is associated with multiple types of cancers, many of which express the viral oncoprotein Latent Membrane Protein 1 (LMP1). LMP1 contributes to both epithelial and B-cell transformation. Although metabolism reprogramming is a cancer hallmark, much remains to be learned about how LMP1 alters lymphocyte oncometabolism. To gain insights into key B-cell metabolic pathways subverted by LMP1, we performed systematic metabolomic analyses on B cells with conditional LMP1 expression. This approach highlighted that LMP highly induces de novo purine biosynthesis, with xanthosine-5-P (XMP) as one of the most highly LMP1-upregulated metabolites. Consequently, IMPDH inhibition by mycophenolic acid (MPA) triggered death of LMP1-expressing EBV-transformed lymphoblastoid cell lines (LCL), a key model for EBV-driven immunoblastic lymphomas. Whereas MPA instead caused growth arrest of Burkitt lymphoma cells with the EBV latency I program, conditional LMP1 expression triggered their death, and this phenotype was rescuable by guanosine triphosphate (GTP) supplementation, implicating LMP1 as a key driver of B-cell GTP biosynthesis. Although both IMPDH isozymes are expressed in LCLs, only IMPDH2 was critical for LCL survival, whereas both contributed to proliferation of Burkitt cells with the EBV latency I program. Both LMP1 C-terminal cytoplasmic tail domains critical for primary human B-cell transformation were important for XMP production, and each contributed to LMP1-driven Burkitt cell sensitivity to MPA. Metabolomic analyses further highlighted roles of NF-kB, mitogen activated kinase, and protein kinase C downstream of LMP1 in support of XMP abundance. Of these, only protein kinase C activity was important for supporting GTP levels in LMP1 expressing Burkitt cells. MPA also de-repressed EBV lytic antigens, including LMP1 itself in latency I Burkitt cells, highlighting crosstalk between the purine biosynthesis pathway and the EBV epigenome. These results suggest novel oncometabolism-based therapeutic approaches to LMP1-driven lymphomas.

Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells, as well as of Kaposi’s sarcoma-associated herpesvirus in B cells, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency. Here, Murray-Nerger et al use a genome-wide CRISPR/Cas9 screen to show that the nuclear protein SFPQ suppresses lytic reactivation of Epstein-Barr virus by promoting the expression and accumulation of linker histone H1 on the viral genome.

Epstein-Barr virus (EBV) uses latency programs to colonize the memory B-cell reservoir, and each program is associated with human malignancies. However, knowledge remains incomplete of epigenetic mechanisms that maintain the highly restricted latency I program, present in memory and Burkitt lymphoma cells, in which EBNA1 is the only EBV-encoded protein expressed. Given increasing appreciation that higher order chromatin architecture is an important determinant of viral and host gene expression, we investigated roles of Wings Apart-Like Protein Homolog (WAPL), a host factor that unloads cohesin to control DNA loop size and that was discovered as an EBNA2-associated protein. WAPL knockout (KO) in Burkitt cells de-repressed LMP1 and LMP2A expression, but not other EBV oncogenes, to yield a viral program reminiscent of EBV latency II, which is rarely observed in B-cells. WAPL KO also increased LMP1/2A levels in latency III lymphoblastoid cells. WAPL KO altered EBV genome architecture, triggering formation of DNA loops between the LMP promoter region and the EBV origins of lytic replication ( oriLyt ). Hi-C analysis further demonstrated that WAPL KO reprogrammed EBV genomic DNA looping. LMP1 and LMP2A de-repression correlated with decreased histone repressive marks at their promoters. We propose that EBV coopts WAPL to negatively regulate latent membrane protein expression to maintain Burkitt latency I.

Microvascular injury is considered an initial event in the pathogenesis of scleroderma and endothelial cells are suspected of being the target of the autoimmune process seen in the disease. EBV has long been proposed as a trigger for autoimmune diseases, including scleroderma. Nevertheless, its contribution to the pathogenic process remains poorly understood. In this study, we report that EBV lytic antigens are detected in scleroderma dermal vessels, suggesting that endothelial cells might represent a target for EBV infection in scleroderma skin. We show that EBV DNA load is remarkably increased in peripheral blood, plasma and circulating monocytes from scleroderma patients compared to healthy EBV carriers, and that monocytes represent the prominent subsets of EBV-infected cells in scleroderma. Given that monocytes have the capacity to adhere to the endothelium, we then investigated whether monocyte-associated EBV could infect primary human endothelial cells. We demonstrated that endothelial cells are infectable by EBV, using human monocytes bound to recombinant EBV as a shuttle, even though cell-free virus failed to infect them. We show that EBV induces activation of TLR9 innate immune response and markers of vascular injury in infected endothelial cells and that up-regulation is associated with the expression of EBV lytic genes in infected cells. EBV innate immune modulation suggests a novel mechanism mediating inflammation, by which EBV triggers endothelial cell and vascular injury in scleroderma. In addition, our data point to up-regulation of EBV DNA loads as potential biomarker in developing vasculopathy in scleroderma. These findings provide the framework for the development of novel therapeutic interventions to shift the scleroderma treatment paradigm towards antiviral therapies.