Epstein-Barr virus (EBV) is a human herpesvirus hiding in a latent form in memory B cells in the majority of the world population. Although, primary EBV infection is asymptomatic or causes a self-limiting disease, infectious mononucleosis, the virus is associated with a wide variety of neoplasms developing in immunosuppressed or immunodeficient individuals, but also in patients with an apparently intact immune system. In memory B cells, tumor cells, and lymphoblastoid cell lines (LCLs, transformed by EBV in vitro) the expression of the viral genes is highly restricted. There is no virus production (lytic viral replication associated with the expression of all viral genes) in tight latency. The expression of latent viral oncogenes and RNAs is under a strict epigenetic control via DNA methylation and histone modifications that results either in a complete silencing of the EBV genome in memory B cells, or in a cell-type dependent usage of latent promoters in tumor cells, germinal center B cells, and LCLs. Both the latent and lytic EBV proteins are potent immunogens and elicit vigorous B- and T-cell responses. In immunosuppressed and immunodeficient patients, or in individuals with a functional defect of EBV-specific T cells, lytic EBV replication is regularly activated and an increased viral load can be detected in the blood. Enhanced lytic replication results in new infection events and EBV-associated transformation events, and seems to be a risk factor both for malignant transformation and the development of autoimmune diseases. One may speculate that an increased load or altered presentation of a limited set of lytic or latent EBV proteins that cross-react with cellular antigens triggers and perpetuates the pathogenic processes that result in multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis. In addition, in SLE patients EBV may cause defects of B-cell tolerance checkpoints because latent membrane protein 1, an EBV-encoded viral oncoprotein can induce BAFF, a B-cell activating factor that rescues self-reactive B cells and induces a lupus-like autoimmune disease in transgenic mice.

The protein product of the CHI3L1 gene, human cartilage 39-kDa glycoprotein (HC-gp39), is a tissue-restricted, chitin-binding lectin and member of glycosyl hydrolase family 18. In contrast to many other monocyte/macrophage markers, its expression is absent in monocytes and strongly induced during late stages of human macrophage differentiation. To gain insights into the molecular mechanisms underlying its cell typerestricted and maturation-associated expression in macrophages, we initiated a detailed study of the proximal HC-gp39 promoter. Deletion analysis of reporter constructs in macrophage-like THP-1 cells localized a region directing high levels of macrophage-specific reporter gene expression to ϳ300 bp adjacent to the major transcriptional start site. The promoter sequence contained consensus binding sites for several known factors, and specific binding of nuclear PU.1, Sp1, Sp3, USF, AML-1, and C/EBP proteins was detectable in gel shift assays. In vivo footprinting assays with dimethyl sulfate demonstrate that the protection of corresponding sequences was enhanced in macrophages compared with monocytes. Mutational analysis of transcription factor binding sites indicated a predominant role for a single Sp1 binding site in regulating HC-gp39 promoter activity. In addition, gel shift assays using nuclear extracts of monocytes and macrophages demonstrated that the binding of nuclear Sp1, but not Sp3, markedly increases during macrophage differentiation. Our results further highlight the important role of Sp1 in macrophage gene regulation.

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Objective The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti-SARS-CoV-2 antibody levels. Methods In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA-and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. Results The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R 2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. Conclusions With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination.

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Two sequences required for activity of the Epstein-Barr virus BART RNA promoter in transfection assays have been identified by site-directed mutagenesis. One contains a consensus AP-1 site; the other has some similarity to Ets and Stat consensus binding sites. Candidate sequences were suggested by mapping a region of unmethylated DNA in EBV around the BART promoter followed by in vivo footprinting the promoter in the C666-1 nasopharyngeal carcinoma cell line, which expresses BART RNAs. The data are presented in the context of a revised EBV DNA sequence, known as EBV wt, that is proposed as a future standard sequence for EBV.

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Epstein-Barr viral (EBV) latency-associated promotersEpstein-Barr virus (EBV) infection is the cause of infectious mononucleosis and is most closely associated with tumor diseases Burkitt's lymphoma (BL) and nasopharyngeal carcinoma. EBV infection of human B lymphocytes in vitro results in B-cell proliferation and transformation into continuously growing lymphoblastoid cell lines (LCL) (for a review, see reference 42). In latently infected cells, viral genomes are maintained as multiple circular episomal copies which are replicated once per cell cycle (2, 103). Several classes of latency have been described depending on the gene expression pattern (41,77,78). In strict type I latency, represented by BL cells, viral gene expression is restricted to the two RNA polymerase III-transcribed EBER RNA genes and the EBNA1 gene (78) that is transcribed from the Q promoter (Qp) (68). The EBNA1 protein is required for the maintenance of the viral plasmid in dividing cells (45,58). In type III latency, in addition to the EBERs, EBNA-LP, -2, -3A, -3B, -3C, and -1 are expressed from the C promoter (Cp) (6), whereas LMP-1 and -2B are expressed from the bidirectional LMP1 promoter (46), and a larger splice variant of LMP-2, LMP-2A, is expressed from the TP1 promoter (36). Qp generally is supposed to be silent in type III latency (82, 105), although there is also a different view (93). Among the viral proteins expressed in latency type III, EBNA2 plays a central role in switching EBNA transcription from Wp to Cp (W to C switch) (102,104) and in the establishment and maintenance of B-cell transformation (11, 28), as EBNA2 transcriptionally activates the expression of the six nuclear antigens from the C promoter (Cp) and the membrane proteins LMP-1 and -2B from the LMP1 promoter (LMP1p), LMP-2A from the TP1 promoter, and a number of cellular proteins associated with the LCL phenotype (1,12,18,39,44,72,76,90,95,98,99,100,101,102,104,110,111). A crucial mechanism involved in the silencing of Cp and LMP1p in type I latency has been shown to be methylation of CpG dinucleotides (3,15,35,54,60,61,70,73,74,75,84,91,94). In LCL, the EBV genome is mostly free of CpG methylation, whereas in BL cells, EBV genomes are highly methylated. An essential step in understanding the differences between latency types I and III is to elucidate the patterns of methylation and in vivo protein binding of the latency promoters of EBV at nucleotide resolution. Therefore, we decided to examine Qp, Cp, and LMP1p in cells of both latency types.(The contributions of Daniel Salamon to this work were made in partial fulfillment of the requirements for a Ph.D. from Semmelweis University, Budapest, Hungary.) MATERIALS AND METHODSCell lines and tissue culture. LCL 721 is a B95-8-transformed LCL with type III phenotype (40,52,57). Rael (15,43,61) is a group I BL cell line. Mutu BLI-C1216 is a subclone of the BL line Mutu, representative of latency type I (27). Mutu BLIII-C199 is a subclone of the BL line Mutu, representative of latency type III (27). Raji cells expre...