Epstein-Barr Virus Polypeptides Research Articles

Survey of Epstein Barr Virus (EBV) Immunogenic Proteins and their Epitopes: Implications for Vaccine Preparation

The Epstein-Barr virus (Human herpesvirus 4) encodes approximately 80 proteins, from which 15 possess at least 90 antigenic epitopes. Many of them stimulate the T cell receptors (TCR), but a few interact with the B cell receptors (BCR). Activation of B-cells and subsequent antibody production has not only been related to at least 3 envelope glycoproteins (mostly gp350) but also to latency associated membrane proteins (LMPs). The majority of EBV epitopes (over 80) inducing either cytotoxic and/or helper T lymphocytes were located on non-structural and/or latency associated polypeptides. The former interaction mediated by CD8plus/T cells is restricted by the HLA I molecules, predominantly of HLA-A subclass. In acute infectious mononucleosis (IM) patients (about 40 %) a considerable proportion of HLA B8 restricted CTL reactivity is directed against a single peptide (RAKFKQLL) of transactivator protein BZLF1/Zta. The EBV vaccines designed so far fall into two categories: those preventing any kind of infection (including prophylaxis of EBVassociated malignancies) and those designed for therapeutic purposes (to be used in subjects already infected). Preventive vaccines protecting against acute disease (such as IM) contain, as a rule, the gp350 polypeptide(s) encoded by the BLLF1 gene. Vaccines destined for tumor prevention rather consist of peptides derived from latency associated nuclear proteins (EBNA 2, 3 and 6) and/or from oncogenic latent membrane proteins (LMP1/LMP2a). Whereas the former generates antibodies preventing virus entry, the latter would potentiate the cell mediated response. In addition to recently described and purified individual recombinant immunogenic EBV polypeptides and/or their mixes, new perspectives were opened by construction of random overlapping strongly immunogenic scrambled polypeptide(s). Further novel approaches are based on carefully selected antigenic peptides (oligopeptides) coming from both, structural as well as non-structural or latencyassociated proteins bound to suitable carriers. Any constructs based on latency-associated proteins might be useful either for immunoprophylactic therapy following bone marrow and/or heart transplantations or for the prevention of EBVrelated tumors such as lymphomas and nasopharyngeal carcinoma. Due to the growing importance of the selected immunogenic epitopes as future vaccine components, at least the half of them has been patented not only as the natural amino acid sequence but also in different variations.

Epstein-Barr Viral Antigens Used in the Diagnosis of Nasopharyngeal Carcinoma.

The major antigen complexes of Epstein-Barr virus (EBV) include the latent infectious proteins, early antigens, membrane antigens and viral capsid antigens. The various polypeptides within each antigen complex have been identified and isolated through gene-cloning technology. These polypeptides are exploited to be used as serological markers for the diagnosis of nasopharyngeal carcinoma (NPC) through enzyme-linked immunosorbent assay. This paper reviews the recent studies on the profile of antibodies in patients with NPC towards these EBV polypeptides of each antigen complex. The sensitivity and specificity of each polypeptide when used as serological markers to NPC patients' sera are summarized. Copyright 1996 S. Karger AG, Basel

Topoisomerase I and II activities are required for Epstein-Barr virus replication.

The roles of topoisomerases I and II in Epstein-Barr virus (EBV) replication were investigated using Raji cells infected with EBV. The topoisomerase II inhibitor ellipticine inhibited the synthesis of EBV polypeptides at concentrations which did not affect total protein synthesis. Slot blot analysis of total cellular DNA showed that camptothecin and ellipticine inhibited replication of progeny EBV DNA in superinfected Raji cells at concentrations which did not inhibit synthesis of EBV early polypeptides prerequisite for EBV DNA replication. Analysis of the structure of EBV DNA termini demonstrated that both inhibitors affected the replicating EBV DNA. Gardella gel electrophoresis showed that both inhibitors affected the formation of the linear form of EBV DNA. However, restriction analysis of EBV DNA in superinfected Raji cells demonstrated that both inhibitors degraded neither endogenous nor exogenous EBV DNA. Cell viability was not affected by either inhibitor at the concentrations tested. These findings suggest that topoisomerase II is required for expression of the EBV genome and that both topoisomerases I and II are involved in replication of the EBV genome during the lytic phase of the life cycle. The effects of topoisomerase inhibitors on the circular form of EBV DNA during virus replication are discussed.

Stress and the memory T-cell response to the Epstein-Barr virus in healthy medical students.

This study investigated the memory T-cell proliferative response to several early and late Epstein-Barr virus (EBV) polypeptides. Blood samples were collected twice, 1 month before a 3-day block of examinations and again on the last day of the exam series. Ss were 25 healthy, EBV seropositive medical students. The proliferative response to 5 of the 6 EBV polypeptides significantly decreased during examinations. In addition, Ss high (above the median) in seeking support, as measured by the COPE, had lower proliferative responses to 3 EBV polypeptides (p17, p52/50, and p85), as well as higher levels of antibody to EBV virus capsid antigen. The data provide further evidence that psychological stress can modulate the cellular immune response to latent EBV.

Neutralization of Epstein-Barr virus-induced ribonucleotide reductase with antibody to the major restricted early antigen polypeptide

A previous study had established a relationship between the major polypeptide of the Epstein-Barr Virus (EBV)-induced restricted (R) component of the early antigen (EA) complex and the large subunit of ribonucleotide reductase. This association was confirmed in this study by the observation that a monoclonal antibody prepared against the 85-kDa R component reacted in immunoblotting with the protein product (molecular weight approximately 93 kDa) encoded by the Xbal fragment of the B-95-8 strain of EBV. This fragment encodes for both the small and large subunits of this virus-induced enzyme. This size of the reactive protein indicated that it was the large subunit. Direct evidence that the 85-kDa EA-R component was associated with the viral-induced ribonucleotide reductase was provided by enzyme inhibition studies. Two monoclonal antibodies prepared against the tertiary structure of the 85-kDa EA-R antigen significantly inhibited the in vitro activity of the viral-specified enzyme but had no effect on ribonucleotide reductase activity derived from two different cellular preparations. A monoclonal antibody prepared against the denatured form of this antigen was ineffective in neutralizing the virus-specific ribonucleotide reductase as were antibodies to different EBV polypeptides. These results therefore conclusively demonstrate that the 85-kDa EA-R polypeptide is associated with the large subunit of this viral-specified enzyme and further suggest that the active site of the enzyme is associated with the tertiary structure of the large subunit.

Amplification of Epstein-Barr virus (EBV) DNA by superinfection with a strain of EBV derived from nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) from a nasopharyngeal carcinoma (NPC) hybrid cell line (NPC-KT) lacking defective viral DNA molecules superinfected Raji cells and induced EBV early antigens (EA), as did virus from P3HR-1 cells, which contained defective molecules. The EBV polypeptides induced by NPC-KT appeared to be identical to those induced by P3HR-1 virus. The ability of NPC-KT virus to induce EA was enhanced more than 10-fold by treatment of superinfected cells with dimethyl sulfoxide; however, dimethyl sulfoxide treatment did not enhance superinfection by P3HR-1 virus. After infection, DNA synthesis of both the superinfecting NPC-KT virus and the resident Raji viral genome was induced. In addition to amplified Raji EBV episomal DNA, a fused terminal fragment of NPC-KT viral DNA was detected. The detection of fused terminal DNA fragments suggests that the superinfecting virion DNA either circularizes or polymerizes after superinfection and is possibly amplified through circular or concatenated replicative intermediates.

Epstein-Barr virus polypeptides: identification of early proteins and their synthesis and glycosylation.

We have identified at least six early polypeptides induced by Epstein-Barr virus in cells or under conditions which are nonpermissive for Epstein-Barr virus DNA replication ranging in molecular weight from 140,000 to 26,000.

Early DNA-binding polypeptides of Epstein-Barr virus

The kinetics of Epstein-Barr virus (EBV)-specific protein synthesis has been studied after induction of P3HR-1 cells with n-butyrate. The majority of the EBV polypeptides became detectable on SDS-polyacrylamide gel electrophoresis between 15 and 18 hr after addition of the drug. Overall viral protein synthesis was at maximum after about 24 hr. Some polypeptides appeared significantly earlier (152K, 55K, 51K) while others were synthesized during a limited period of the cycle only (65K). At least four of the polypeptides detected (152K, 134K, 55K, 51K) bound to DNA cellulose columns. The EBV specificity of the polypeptides was confirmed by immunoprecipitation and by two-dimensional gel analysis. The 152K polypeptide had a p I around 8 whereas the other polypeptides varied between basic and acidic p I. The DNA binding proteins isolated by DNA cellulose chromatography represent a subclass of the virus-specific early proteins that can be immunoprecipitated from the total cell extract.

Epstein-Barr virus polypeptides: effect of inhibition of viral DNA replication on their synthesis.

Epstein-Barr virus polypeptides: effect of inhibition of viral DNA replication on their synthesis.

Epstein-Barr Virus Polypeptides: Identification of Early Proteins and Their Synthesis and Glycosylation

Epstein-Barr Virus Polypeptides: Identification of Early Proteins and Their Synthesis and Glycosylation

Epstein-Barr virus polypeptides: effect of inhibition of viral DNA replication on their synthesis.

After Epstein-Barr virus superinfection of the human lymphoblastoid cell line Raji, a Burkitt lymphoma-derived line that contains Epstein-Barr virus genomes in an episomal form, at least 40 polypeptides could be resolved by polyacrylamide gel electrophoresis. Eleven of the 40 polypeptides were immunoprecipitable by early antigen+/viral capsid antigen+ antiserum. The polypeptides could be divided into six classes, immediate-early, early, intermediate, late, very late, and persistent, depending upon the time of synthesis. Ten of the 40 polypeptides appeared to preexist before superinfection and persisted despite general cessation of host protein synthesis; none of the persistent proteins was immunoprecipitated by the Epstein-Barr virus antibody-containing serum. When viral DNA replication was blocked by a variety of inhibitors of DNA synthesis, a number of different patterns of polypeptide synthesis could be detected. The synthesis of six polypeptides was blocked by the most virus-specific inhibitors, acyclovir and phosphonoacetic acid. Additionally, in the presence of 1-beta-D-arabinofuranosylcytosine, 1-beta-D-arabinofuranosyladenine, and methotrexate, seven polypeptides showed oversynthesis.

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.