Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation.

Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism

Genetic Analysis of Human Traits In Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype–phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance—i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.Molecular and Cellular Biolog

The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses

Genetic Analysis of Human Traits In Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype–phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance—i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding

LMP1 Transmembrane Domain 1 and 2 (TM1-2) FWLY Mediates Intermolecular Interactions with TM3-6 To Activate NF-κB

The Epstein-Barr virus oncoprotein LMP1 has six transmembrane domains (TMs) that enable intermolecular aggregation and constitutive signaling through two C-terminal cytosolic domains. Expression of both TMs 1 and 2 without the C terminus (TM1-2ΔC) and TMs 3 to 6 fused to the C terminus (TM3-6) results in partial association, which is substantially decreased by TM1 F(38)WLY(41) mutation to A(38)ALA(41). We now investigate whether TM1-2ΔC can functionally interact with TM3-6. TM1-2ΔC induced TM3-6 to mediate NF-κB activation at 59% of LMP1 levels, and the effect was dependent on TM1-2 F(38)WLY(41). TM1-2ΔC even induced TM3-4 C terminus-mediated NF-κB activation to 44% of LMP1 levels. Surprisingly, this effect was TM1 F(38)WLY(41) independent, indicative of a role for TMs 5 and 6 in TM1 F(38)WLY(41) effects. TM3 W(98) was also important for TM1-2ΔC induction of TM3-6-mediated NF-κB activation, for association, and for TM1 F(38)WLY(41) dependence on C-terminal NF-κB activation. These data support models in which the TM1 F(38)WLY(41) effects are at least partially dependent on TM3 W(98) and a residue(s) in TMs 5 and 6

Virus and Cell RNAs Expressed during Epstein-Barr Virus Replication

Changes in Epstein-Barr virus (EBV) and cell RNA levels were assayed following immunoglobulin G (IgG) cross-linking-induced replication in latency 1-infected Akata Burkitt B lymphoblasts. EBV replication as assayed by membrane gp350 expression was ∼5% before IgG cross-linking and increased to more than 50% 48 h after induction. Seventy-two hours after IgG cross-linking, gp350-positive cells excluded propidium iodide as well as gp350-negative cells. EBV RNA levels changed temporally in parallel with previously defined sensitivity to inhibitors of protein or viral DNA synthesis. BZLF1 immediate-early RNA levels doubled by 2 h and reached a peak at 4 h, whereas BMLF1 doubled by 4 h with a peak at 8 h, and BRLF1 doubled by 8 h with peak at 12 h. Early RNAs peaked at 8 to 12 h, and late RNAs peaked at 24 h. Hybridization to intergenic sequences resulted in evidence for new EBV RNAs. Surprisingly, latency III (LTIII) RNAs for LMP1, LMP2, EBNALP, EBNA2, EBNA3A, EBNA3C, and BARTs were detected at 8 to 12 h and reached maxima at 24 to 48 h. EBNA2 and LMP1 were at full LTIII levels by 48 h and localized to gp350-positive cells. Thus, LTIII expression is a characteristic of late EBV replication in both B lymphoblasts and epithelial cells in immune-comprised people (J. Webster-Cyriaque, J. Middeldorp, and N. Raab-Traub, J. Virol. 74:7610-7618, 2000). EBV replication significantly altered levels of 401 Akata cell RNAs, of which 122 RNAs changed twofold or more relative to uninfected Akata cells. Mitogen-activated protein kinase levels were significantly affected. Late expression of LTIII was associated with induction of NF-κB responsive genes including IκBα and A20. The exclusion of propidium, expression of EBV LTIII RNAs and proteins, and up-regulation of specific cell RNAs are indicative of vital cell function late in EBV replication

Construction of the LMP1Δ Bacterial artificial chromosome.

<p>(A) Schematic of wildtype and resulting LMP1Δ viruses. EBV coordinates according to B958 numbering. Primers for PCR screening indicated. (B) PCR across the LMP1 loci of the indicated BAC. (C) BAMH1 digest indicates the wt and LMP1Δ BAC genomes have no gross deletion. A unique 8.2 kB fragment (*) corresponds to the expected size of the BAM N fragment harboring the LMP1 deletion.</p

LMP1Δ virus shows no defect in egress, encapsidation or infectivity.

<p>(A) 3 representative Transmission Electron Micrographs of HEK293 cells producing LMP1Δ virus, black bars = 500 nm. (B) LMP1Δ virus shows no defect in egress from HEK293 cells. Quantization of intracellular and extracellular EBV DNA by qPCR for BALF5 (not normalized to gp350). Ratio between the two is presented (n>5, standard deviation is shown). (C) Extracellular LMP1Δ virus is encapsidated normally. Extracellular virions were digested with or without DNAse 1 and quantitated by BALF5 qPCR. BAC DNA produced in E. Coli served as a DNAse sensitive control (n = 2). (D) LMP1Δ virus is equally infectious as wt virus. BJAB was infected and colony formation after puromycin selection was determined at 4–6weeks post infection. BJAB infectious units are plotted as a function of EBV BALF5 copy number in the supernatants (n = 3). The relationship between EBV copy number and BJAB infectious units is linear, R² = .57. Open symbols, LMP1 was transfected with Zta, closed symbols, Zta alone.</p

LMP1Δ BAC replicates to wildtype levels in HEK293 cells.

<p>(A) Western blot analysis of 293 cells with the indicated BAC 4 days after the virus was induced to replicate with Zta or Zta and LMP1 expression. Rta, BMRF1 and lytic protein expression was induced to the same extent in wt and LMP1Δ cells. LMP1 transcomplementation inhibited Rta expression. (B) Zta induces virus DNA replication in wt and LMP1Δ HEK293 cells, Gardella gel analysis, linear genomes marked by black bar. (C) Zta-induced replication is unaffected by LMP1 deletion; .EBV (BALF5) per GAPDH copies of intracellular virus was normalized to the percentage of gp350+ cells.</p