Carboxyl Region Research Articles

The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly.

Mitochondrial bc 1 complex from yeast has 10 subunits, but only cytochrome b (Cytb) subunit is encoded in the mitochondrial genome. Cytb has eight transmembrane helices containing two hemes b for electron transfer. Cbp3 and Cbp6 assist Cytb synthesis, and together with Cbp4 induce Cytb hemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cytb synthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Because Qcr7 resides near the Cytb carboxyl region, we wondered whether this region is important for Cytb synthesis/assembly. Although deletion of the Cytb C-region did not abrogate Cytb synthesis, the assembly-feedback regulation was lost, so Cytb synthesis was normal even if Qcr7 was missing. Mutants lacking the Cytb C-terminus were non-respiratory because of the absence of fully assembled bc 1 complex. By performing complexome profiling, we showed the existence of aberrant early-stage subassemblies in the mutant. In this work, we demonstrate that the C-terminal region of Cytb is critical for regulation of Cytb synthesis and bc 1 complex assembly.

On the comparison between differential vibrational spectroscopy spectra and theoretical data in the carboxyl region of photosystem II.

Understanding the structural modification experienced by the Mn4 CaO5 oxygen-evolving complex of photosystem II along the Kok-Joliot's cycle has been a challenge for both theory and experiments since many decades. In particular, differential infrared spectroscopy was extensively used to probe the surroundings of the reaction center, to catch spectral changes between different S-states along the catalytic cycle. Because of the complexity of the signals, only a limited quantity of identified peaks have been assigned so far, also because of the difficulty of a direct comparison with theoretical calculations. In the present work, we critically reconsider the comparison between differential vibrational spectroscopy and theoretical calculations performed on the structural models of the photosystem II active site and an inorganic structural mimic. Several factors are currently limiting the reliability of a quantitative comparison, such as intrinsic errors associated to theoretical methods, and most of all, the uncertainty attributed to the lack of knowledge about the localization of the underlying structural changes. Critical points in this comparison are extensively discussed. Comparing several computational data of differential S2 /S1 infrared spectroscopy, we have identified weak and strong points in their interpretation when compared with experimental spectra.

A novel mutation in COL1A2 leads to osteogenesis imperfecta/Ehlers-Danlos overlap syndrome with brachydactyly

Osteogenesis imperfecta (OI) is mainly characterized by bone fragility and Ehlers-Danlos syndrome (EDS) by connective tissue defects. Mutations in COL1A1 or COL1A2 can lead to both syndromes. OI/EDS overlap syndrome is mostly caused by helical mutations near the amino-proteinase cleavage site of type I procollagen. In this study, we identified a Thai patient having OI type III, EDS, brachydactyly, and dentinogenesis imperfecta. His dentition showed delayed eruption, early exfoliation, and severe malocclusion. For the first time, ultrastructural analysis of the tooth affected with OI/EDS showed that the tooth had enamel inversion, bone-like dentin, loss of dentinal tubules, and reduction in hardness and elasticity, suggesting severe developmental disturbance. These severe dental defects have never been reported in OI or EDS. Exome sequencing identified a novel de novo heterozygous glycine substitution, c.3296G > A, p.Gly1099Glu, in exon 49 of COL1A2. Three patients with mutations in the exon 49 of COL1A2 were previously reported to have OI with brachydactyly and intracranial hemorrhage. Notably, two of these three patients did not show hyperextensible joints and hypermobile skin, while our patient at the age of 5 years had not developed intracranial hemorrhage. Here, we demonstrate that the novel glycine substitution in the carboxyl region of alpha2(I) collagen triple helix leads to OI/EDS with brachydactyly and severe tooth defects, expanding the genotypic and phenotypic spectra of OI/EDS overlap syndrome.

Regulation of stringent factor by branched-chain amino acids

When faced with amino acid starvation, prokaryotic cells induce a stringent response that modulates their physiology. The stringent response is manifested by production of signaling molecules guanosine 5'-diphosphate,3'-diphosphate (ppGpp) and guanosine 5'-triphosphate,3'-diphosphate (pppGpp) that are also called alarmones. In many species, alarmone levels are regulated by a multidomain bifunctional alarmone synthetase/hydrolase called Rel. In this enzyme, there is an ACT domain at the carboxyl region that has an unknown function; however, similar ACT domains are present in other enzymes that have roles in controlling amino acid metabolism. In many cases, these other ACT domains have been shown to allosterically regulate enzyme activity through the binding of amino acids. Here, we show that the ACT domain present in the Rhodobacter capsulatus Rel alarmone synthetase/hydrolase binds branched-chain amino acids valine and isoleucine. We further show that the binding of these amino acids stimulates alarmone hydrolase activity both in vitro and in vivo. Furthermore, we found that the ACT domain present in Rel proteins from many diverse species also binds branched-chain amino acids. These results indicate that the cellular concentration of amino acids can directly affect Rel alarmone synthetase/hydrolase activity, thus adding another layer of control to current models of cellular control of the stringent response.

Antibodies that bind to an EBV derived peptide mimic of dsDNA, have pathogenic potential

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of antibodies to double stranded DNA (dsDNA) that can deposit in the kidney leading to nephritis. Our laboratory has previously been studying the association of SLE with the Epstein Barr Virus (EBV). We generated two monoclonal antibodies designated 3D4 and 16D2, that bind to a peptide (PFM-15) in the carboxyl region of a major nuclear protein of EBV (EBNA-1) and cross-react with dsDNA. In the present study, we demonstrate that both of these monoclonal antibodies can bind to fixed preparations of rat glomeruli suggesting pathogenic potential. Immunostaining is dramatically reduced when glomeruli are pre-treated with DNase indicating that these antibodies recognize chromatin associated structures. In addition, the PFM-15 peptide inhibits 3D4 and 16D2 from binding to glomeruli and inhibits both antibodies from binding to dsDNA coated plates by ELISA. This suggests that PFM-15 can act as a molecular mimic of dsDNA. Our results demonstrate that antibodies that recognize a viral peptide and cross-react with dsDNA can bind to glomeruli and therefore have pathogenic potential. This study further supports an association between EBV and SLE and supports the role of molecular mimicry in autoimmune disease.

Different phosphorylation patterns regulate α1D-adrenoceptor signaling and desensitization

Human α1D-adrenoceptors (α1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α1D-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/β, phosphorylate α1D-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α1-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.

Mapping an epitope in EBNA-1 that is recognized by monoclonal antibodies to EBNA-1 that cross-react with dsDNA.

IntroductionThe Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen‐I (EBNA‐1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA‐1, 3D4, and demonstrated that it cross‐reacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA‐1. The aim of the present study was to characterize another antibody to EBNA‐1 that cross‐reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA‐1 that is recognized by these cross‐reactive antibodies.MethodsWe generated an IgM MAb to EBNA‐1, 16D2, from EBNA‐1 injected mice and demonstrated by ELISA that it cross‐reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA‐1 recognized by these MAbs, we examined their binding by ELISA to 15 overlapping peptides spanning the 148 amino acid domain.ResultsSequence analysis revealed that 3D4 and 16D2 utilize different VH and VL genes but identical JH and Jk regions with minimal junctional diversity. This accounts for similarities in their CDR3 regions and may explain their similar dual binding specificity. Epitope mapping revealed 3D4 and 16D2 bind the same peptide in the VBS. Based on the crystal structure of EBNA‐1, we observed that this peptide resides at the base of an exposed proline rich loop in EBNA‐1.ConclusionWe have demonstrated that two MAbs that bind EBNA‐1 and cross‐react with dsDNA, recognize the same peptide in the VBS. This peptide may serve as a mimetope for dsDNA and may be of diagnostic and therapeutic value in SLE.

Synthesis, structure-activity relationship and biological evaluation of novel nitrogen mustard sophoridinic acid derivatives as potential anticancer agents.

A series of novel nitrogen mustard sophoridinic acid derivatives were designed, synthesized and evaluated for their cytotoxicity. Of the newly synthesized compounds, compound 6 exhibited a potent effect against hepatocellular carcinoma in vitro and in vivo. SAR analysis indicated that introduction of a nitrogen mustard group to the structure of sophoridinic acid significantly enhance the antitumor activity. Moreover, molecular docking study exhibited benzyl group introduced to the nitrogen atom at the 12-position and aryl nitrogen mustard group at the 4'-carboxyl region for compound 6 were beneficial for the higher anticancer activity. This work provides useful information for further structural modifications of these compounds and for the synthesis of new, potent antitumor agents.

Mice Deficient in AKAP13 (BRX) Are Osteoporotic and Have Impaired Osteogenesis

Mechanical stimulation is crucial to bone growth and triggers osteogenic differentiation through a process involving Rho and protein kinase A. We previously cloned a gene (AKAP13, aka BRX) encoding a protein kinase A-anchoring protein in the N-terminus, a guanine nucleotide-exchange factor for RhoA in the mid-section, coupled to a carboxyl region that binds to estrogen and glucocorticoid nuclear receptors. Because of the critical role of Rho, estrogen, and glucocorticoids in bone remodeling, we examined the multifunctional role of Akap13. Akap13 was expressed in bone, and mice haploinsufficient for Akap13 (Akap13(+/-)) displayed reduced bone mineral density, reduced bone volume/total volume, and trabecular number, and increased trabecular spacing; resembling the changes observed in osteoporotic bone. Consistent with the osteoporotic phenotype, Colony forming unit-fibroblast numbers were diminished in Akap13(+/-) mice, as were osteoblast numbers and extracellular matrix production when compared to control littermates. Transcripts of Runx2, an essential transcription factor for the osteogenic lineage, and alkaline phosphatase (Alp), an indicator of osteogenic commitment, were both reduced in femora of Akap13(+/-) mice. Knockdown of Akap13 reduced levels of Runx2 and Alp transcripts in immortalized bone marrow stem cells. These findings suggest that Akap13 haploinsufficient mice have a deficiency in early osteogenesis with a corresponding reduction in osteoblast number, but no impairment of mature osteoblast activity.

Water/Alcohol Soluble Poly(3-Alkylthiophene) Derivative Self-Assembly with Fullerene Derivative via Hydrogen Bonding

In this research, a regioregular poly (3-alkylthiophene) derivative had been synthesized via the post-polymerization functionalization, poly (3-hexylamine thiophene), which are soluble in water and water miscible solvents such as methanol, DMSO. The suitable energy level, good thermal stability and water/alcohol solubility of polymers which promising applied in polymer solar cells and processed by water or environmental-friendly solvents. In blend of functionalization polymers and fullerene derivative potassium [6,6]-phenyl-C61-butyrate shows significant UV absorption decay phenomenon and occur precipitates follow with time increase. To further investigate the mechanism of composite, FT-IR analysis and TEM investigate were used, the polymer would be bonded with carboxyl of fullerene derivative via hydrogen bonding in the carboxyl region and TEM images demonstration the mixture that be able to self-assembly a interpenetrating network composite.

Expression of Pneumocystis jirovecii Major Surface Glycoprotein in Saccharomyces cerevisiae

The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis organisms, recombinant Msg localized to the plasma membrane of yeast rather than to the cell wall. Msg expression was targeted to the yeast cell wall by replacing its signal peptide, serine-threonine-rich region, and glycophosphatidylinositol anchor signal region with the signal peptide of cell wall protein α-agglutinin of S. cerevisiae, the serine-threonine-rich region of epithelial adhesin (Epa1) of Candida glabrata, and the carboxyl region of the cell wall protein (Cwp2) of S. cerevisiae, respectively. Immunofluorescence analysis and treatment with β-1,3 glucanase demonstrated that the expressed Msg fusion protein localized to the yeast cell wall. Surface expression of Msg protein resulted in increased adherence of yeast to A549 alveolar epithelial cells. Heterologous expression of Msg in yeast will facilitate studies of the biologic properties of Pneumocystis Msg.

분열효모 핵공단백질인 Nup184의 기능에 필요한 부위 분석 및 SUMO 변성

The Nup188 protein is one of the largest evolutionally conserved nucleoprins (Nups) that compose the inner ring of nuclear pore complex (NPC). The Nup184 protein, fission yeast Schizosaccharomyces pombe ortholog of Nup188p, is required for normal growth and mRNA export in nutrient-rich medium (YES). Here, we identified a carboxyl region (482 to 1628) of Nup184 protein that was enough to complement the defects of both growth and mRNA export when the knock-out mutant was grown in YES medium. This region is also required for localization of GFP-Nup184 fusion to the nuclear periphery. In addition, we found that ORF of Nup184 (predicted 1564 amino-acid protein) registered in S. pombe GeneDB (hosted by Sanger Institute, UK) is 64 amino-acid residues shorter than that predicted by our sequence data. This carboxy-terminal region is necessary for the functions of Nup184p. We further demonstrated that Nup184 protein was conjugated with SUMO in vivo.

Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

Robust hepatitis B virus (HBV) replication is stimulated by the regulatory HBx protein. HBx binds the cellular protein DDB1; however, the importance of this interaction for HBV replication remains unknown. We tested whether HBx binding to DDB1 was required for HBV replication using a plasmid based replication assay in HepG2 cells. Three DDB1 binding-deficient HBx point mutants (HBx69, HBx90/91, HBxR96E) failed to restore wildtype levels of replication from an HBx-deficient plasmid, which established the importance of the HBx-DDB1 interaction for maximal HBV replication. Analysis of overlapping HBx truncation mutants revealed that both the HBx-DDB1 binding domain and the carboxyl region are required for maximal HBV replication both in vitro and in vivo, suggesting the HBx-DDB1 interaction recruits regulatory functions critical for replication. Finally we demonstrate that HBx localizes to the Cul4A-DDB1 complex, and discuss the possible implications for models of HBV replication.

Identification and characterization of Penicillium citrinum VeA and LaeA as global regulators for ML-236B production

In filamentous fungi, production of multiple secondary metabolites is controlled by so-called global regulators. In this study, two genes encoding homologs of VeA and LaeA, representative fungal global regulators, were identified in ML-236B-producing Penicillium citrinum. Disruption of VeA and/or LaeA and complementation clearly demonstrated that both of them played critical roles in ML-236B production by controlling the expression of mlcR, the pathway-specific activator gene for ML-236B biosynthesis. Moreover, sequence analysis revealed that laeA in a mutant strain producing high levels of ML-236B (strain S-1567) possessed a single nucleotide alteration, which resulted in 15 surplus amino acids at the carboxyl terminus of LaeA compared to the LaeA in the wild-type strain (strain SANK18767). Introduction of the mutated laeA into SANK18767 proved that the extended carboxyl region plays a crucial role in the higher production of ML-236B. These results indicated that VeA and LaeA dominantly control the biosynthesis of ML-236B, and the enhanced production in the strain S-1567 is attributable to the mutation in laeA.

Analyses of the Functional Regions of DEAD-Box RNA “Helicases” with Deletion and Chimera Constructs Tested In Vivo and In Vitro

The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found in nearly all organisms and that are involved in virtually all processes involving RNA. They are characterized by two tandemly linked, RecA-like domains that contain 11 conserved motifs and highly variable amino- and carboxy-terminal flanking sequences. For this reason, they are often considered to be modular multi-domain proteins. We tested this by making extensive BLASTs and sequence alignments to elucidate the minimal functional unit in nature. We then used this information to construct chimeras and deletions of six essential yeast proteins that were assayed in vivo. We purified many of the different constructs and characterized their biochemical properties in vitro. We found that sequence elements can only be switched between closely related proteins and that the carboxy-terminal sequences are important for high ATPase and strand displacement activities and for high RNA binding affinity. The amino-terminal elements were often toxic when overexpressed in vivo, and they may play regulatory roles. Both the amino and the carboxyl regions have a high frequency of sequences that are predicted to be intrinsically disordered, indicating that the flanking regions do not form distinct modular domains but probably assume an ordered structure with ligand binding. Finally, the minimal functional unit of the DEAD-box core starts two amino acids before the isolated phenylalanine of the Q motif and extends to about 35 residues beyond motif VI. These experiments provide evidence for how a highly conserved structural domain can be adapted to different cellular needs.

Hepatic Deficiency of Low Density Lipoprotein Receptor-related Protein-1 Reduces High Density Lipoprotein Secretion and Plasma Levels in Mice

The low density lipoprotein receptor-related protein-1 (LRP1) is known to serve as a chylomicron remnant receptor in the liver responsible for the binding and plasma clearance of apolipoprotein E-containing lipoproteins. Previous in vitro studies have provided evidence to suggest that LRP1 expression may also influence high density lipoprotein (HDL) metabolism. The current study showed that liver-specific LRP1 knock-out (hLrp1(-/-)) mice displayed lower fasting plasma HDL cholesterol levels when compared with hLrp1(+/+) mice. Lecithin:cholesterol acyl transferase and hepatic lipase activities in plasma of hLrp1(-/-) mice were comparable with those observed in hLrp1(+/+) mice, indicating that hepatic LRP1 inactivation does not influence plasma HDL remodeling. Plasma clearance of HDL particles and HDL-associated cholesteryl esters was also similar between hLrp1(+/+) and hLrp1(-/-) mice. In contrast, HDL secretion from primary hepatocytes isolated from hLrp1(-/-) mice was significantly reduced when compared with that observed with hLrp1(+/+) hepatocytes. Biotinylation of cell surface proteins revealed decreased surface localization of the ATP-binding cassette, subfamily A, member 1 (ABCA1) protein, but total cellular ABCA1 level was not changed in hLrp1(-/-) hepatocytes. Finally, hLrp1(-/-) hepatocytes displayed reduced binding capacity for extracellular cathepsin D, resulting in lower intracellular cathepsin D content and impairment of prosaposin activation, a process that is required for membrane translocation of ABCA1 to facilitate cholesterol efflux and HDL secretion. Taken together, these results documented that hepatic LRP1 participates in cellular activation of lysosomal enzymes and through this mechanism, indirectly modulates the production and plasma levels of HDL.

Mapping the epitope in Epstein Barr Virus nuclear antigen-1 that elicits cross-reactivity with dsDNA (101.43)

Abstract Several studies have shown an association between Systemic Lupus Erythematosus (SLE) and the Epstein Barr Virus (EBV). Our laboratory recently demonstrated that BALB/c mice injected with the major EBV nuclear antigen, EBNA-1, developed antibodies to EBNA-1 that cross-react with double stranded DNA (dsDNA). We generated monoclonal antibodies (MAbs) to EBNA-1 from EBNA-1 injected mice and demonstrated by anti-dsDNA ELISA, dsDNA affinity columns, and Crithidia luciliae assays, that these antibodies cross-react with dsDNA. Studies are underway to determine the pathogenicity of these MAbs. We have been mapping the epitope in EBNA-1 that serves as a peptide mimotope for dsDNA. We now demonstrate that the mimotope is contained within the carboxyl region of the EBNA-1 protein, between amino acids 459 and 607. This region contains well-defined secondary structure, which suggests that the basis for the cross-reactivity with dsDNA could be due to a conformational epitope in EBNA-1. Studies are in progress to further map this cross-reactive epitope. Identification of this epitope may help in designing diagnostic and therapeutic strategies that can mask the epitope from the immune system following EBV infection and thereby prevent the development of antibodies that cross-react with dsDNA.

Point mutations in the Rpb9-homologous domain of Rpc11 that impair transcription termination by RNA polymerase III

RNA polymerase III recognizes and pauses at its terminator, an oligo(dT) tract in non-template DNA, terminates 3′ oligo(rU) synthesis within this sequence, and releases the RNA. The pol III subunit Rpc11p (C11) mediates RNA 3′–5′ cleavage in the catalytic center of pol III during pausing. The amino and carboxyl regions of C11 are homologous to domains of the pol II subunit Rpb9p, and the pol II elongation and RNA cleavage factor, TFIIS, respectively. We isolated C11 mutants from Schizosaccharomyces pombe that cause pol III to readthrough terminators in vivo. Mutant RNA confirmed the presence of terminator readthrough transcripts. A predominant mutation site, F32, resides in the C11 Rpb9-like domain. Another mutagenic approach confirmed the F32 mutation and also isolated I34 and Y30 mutants. Modeling Y30, F32 and I34 of C11 in available cryoEM pol III structures predicts a hydrophobic patch that may interface with C53/37. Another termination mutant, Rpc2-T455I, appears to reside internally, near the RNA–DNA hybrid. We show that the Rpb9 and TFIIS homologous mutants of C11 reflect distinct activities, that differentially affect terminator recognition and RNA 3′ cleavage. We propose that these C11 domains integrate action at the upper jaw and center of pol III during termination.

Preclinical Vaccine Study of Plasmodium vivax Circumsporozoite Protein Derived-Synthetic Polypeptides Formulated in Montanide ISA 720 and Montanide ISA 51 Adjuvants

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

Antibodies Elicited in Response to EBNA-1 May Cross-React with dsDNA

BackgroundSeveral genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the complete EBNA-1 protein can develop antibodies to double stranded DNA (dsDNA). The present study was undertaken to understand why anti-dsDNA antibodies arise during the immune response to EBNA-1.Methodology/Principal FindingsIn this study, we demonstrated that mouse antibodies elicited in response to EBNA-1 cross-react with dsDNA. First, we showed that adsorption of sera reactive with EBNA-1 and dsDNA, on dsDNA cellulose columns, diminished reactivity with EBNA-1. Next, we generated mononclonal antibodies (MAbs) to EBNA-1 and showed, by several methods, that they also reacted with dsDNA. Examination of two cross-reactive MAbs—3D4, generated in this laboratory, and 0211, a commercial MAb—revealed that 3D4 recognizes the carboxyl region of EBNA-1, while 0211 recognizes both the amino and carboxyl regions. In addition, 0211 binds moderately well to the ribonucleoprotein, Sm, which has been reported by others to elicit a cross-reactive response with EBNA-1, while 3D4 binds only weakly to Sm. This suggests that the epitope in the carboxyl region may be more important for cross-reactivity with dsDNA while the epitope in the amino region may be more important for cross-reactivity with Sm.Conclusions/SignificanceIn conclusion, our results demonstrate that antibodies to the EBNA-1 protein cross-react with dsDNA. This study is significant because it demonstrates a direct link between the viral antigen and the development of anti-dsDNA antibodies, which are the hallmark of SLE. Furthermore, it illustrates the crucial need to identify the epitopes in EBNA-1 responsible for this cross-reactivity so that therapeutic strategies can be designed to mask these regions from the immune system following EBV exposure.