Viral Capsid Antigen Expression Research Articles

Expression of VCA (viral capsid antigen) and EBNA1 (Epstein–Barr‐virus‐encoded nuclear antigen 1) genes of Epstein–Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma

EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results in screening for NPC.

Rapid determination of Epstein–Barr virus latent or lytic infection in single human cells using in situ hybridization

Epstein–Barr (EBV) virus is associated with malignancies such as lymphoma and carcinoma. Infection of cells with EBV may result in either lytic infection with production of viral particles, characterized by the presence of linear DNA forms, or latent infection, characterized by either episomal or integrated DNA forms. To examine whether the different lytic and latent EBV DNA forms can reliably be distinguished in single human cells, in situ hybridization was performed in EBV-positive cell lines. Immunocytochemistry and Southern blot analysis were performed supplementary to in situ hybridization. In latent infection, three in situ hybridization patterns were observed: large-disperse (episomal), small-punctate (integrated) and combined (both), signal types 1, 2 and 3 respectively. These were associated with expression of latent membrane protein 1, but not with Z fragment of Epstein–Barr replication activator or viral capsid antigen. In lytic infection, three additional in situ hybridization patterns were observed: nuclear membrane associated, bubble (filling up the nucleus) and spillover (covering the lysed cells) signals types 4, 5 and 6 respectively. Signal types 4 and 5 were associated with expression of latent membrane protein 1 and Z fragment of Epstein–Barr replication activator but not viral capsid antigen, whereas type 6 was associated with expression of viral capsid antigen only. Southern blot analysis confirmed these results; however, low copy numbers of integrated virus were often missed by Southern blot, confirming that in situ hybridization is more sensitive in determining the presence of all types of EBV DNA. In situ hybridization may prove useful in rapidly screening large series of tissue microarrays and other clinical specimens for the presence of lytic or latent EBV.

Inhibitory effects of novel nucleoside and nucleotide analogues on Epstein-Barr virus replication.

The anti-Epstein-Barr virus (EBV) activity of different classes of compounds was assessed by means of an EBV DNA hybridization assay using a digoxigenin-labelled probe specific for the BamHI W fragment of the EBV genome, as well as by measuring viral capsid antigen (VCA) expression after a 7 day incubation period of P3HR-1 producer cells with the test substances. Acyclovir, ganciclovir, cidofovir and zidovudine were included as reference compounds. Several compounds proved to be potent and selective inhibitors of EBV DNA synthesis and VCA expression. Of the new compounds that were evaluated for their anti-EBV activity, the highest efficacy (lowest EC50) and highest selectivity index (SI) were shown by the purine nucleoside analogue 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S2242) (EC50 0.6 ng/ml; SI 600), the acyclic nucleoside phosphonate analogues 9-(2-phosphono -methoxyethyl)-6-dimethylaminopurine (EC50 1.1 micrograms/ml; SI 91), 9-(2-phosphonomethoxyethyl)-2- amino-6-benzhydrylaminopurine (EC50 1.3 micrograms/ml; SI 29), 7-(2-phosphonomethoxyethyl)-6-dimethyl-aminopurine (EC50 0.8 microgram/ml; SI 56), 9-(R)-(2-phosphonomethoxypropyl)-6-(2-dimethylaminoethyl)-aminopur ine (EC50 0.5 microgram/ml; SI 42), the 2',3'-dideoxythymidine derivative 3'-oximino-2',3'-dideoxythymidine (EC50 1.5 micrograms/ml; SI 65), and 1-(2,3- dideoxy-3-N-hydroxyamino-beta-D-threo-pentafuranyl)pentafuranos yl)thymine (EC50 4.1 micrograms/ml; SI > 24).

Relationship between methylation status and expression of an epstein-barr virus (EBV) capsid antigen gene

The methylation status of the 160 kD viral capsid antigen ( VCA ) gene promoter was determined by hybridization analysis. The semi-permissive marmoset cell line FF41-1 lacked cytosine methylation in approximately three quarters of the VCA promoter CpG dinucleotide residues. In the stringently infected HH514CL16 cell line the same CpG residues were methylated in three quarters of the genomes. 5′deoxy-5′-S-isobutyladenosine (SIBA), a DNA methylase inhibitor, was utilized to disrupt the EBV latent state. As determined by flow cytometry, SIBA treatment significantly increased expression of VCA. The VCA promoter was hypomethylated in VCA-positive FF41-1 cells sorted by flow cytometry. While hypomethylation alone was not sufficient for VCA transcriptional activity, the absence of methylation of VCA promoter CpG dinucleotide residues was associated with expression of VCA.

Inhibition of Epstein-Barr virus (EBV) release from the P3HR-1 Burkitt's lymphoma cell line by a monoclonal antibody against a 200,000 dalton EBV membrane antigen.

In raising murine hybridoma antibodies against Epstein-Barr virus (EBV)-induced membrane antigens (MA), we found one antibody that blocked the release of infectious EBV from cultured P3HR-1 cells. This monoclonal antibody (mAb) recognized a 200 kD, phosphonoacetic acid-sensitive (late) MA, and did not directly neutralize virus without complement. When this mAb was added to 33 degrees C-cultured, spontaneously EBV-producing P3HR-1 cells, the intracellular expression of viral capsid antigen and infectious virus was not inhibited, but the appearance of infectious virus in the culture medium was significantly reduced. The duration of this suppression was dependent upon the concentration of the mAb, an effect being observed to a 1:4 X 10(5) titer of the ascites mAb preparation. A more acute effect of suppression of EBV release was observed in a second model of 12-o-tetradecanoyl phorbol-13-acetate and n-butyrate induction of EBV in 37 degrees C-cultured P3HR-1 cells. Again, intracellular infectious virus production was not inhibited, but the level of infectious virus in the culture medium was significantly reduced as early as 1 and 2 d of culture with antibody. This effect was reversed within 31 h after replacement of mAb-containing medium with fresh medium. This description of antibody-mediated inhibition of EBV release might lead to the characterization of another form of immune defense for the control of EBV infections.

Effect of (E)-5-(2-Bromovinyl)-2'-deoxyuridine on the Growth and Viral Capsid Antigen Expression of Epstein-Barr Virus-Associated Tumor (B-95-8) Cells Transplanted to Nude Mice

When persistently Epstein-Barr virus (EBV)-infected lymphoblastoid (B-95-8) cells were transplanted subcutaneously or intracerebrally to nude mice of either BALB/c or NIH background, tumors developed, and the tumor cells spontaneously expressed viral capsid antigen (VCA). This model was used to evaluate the in vivo anti-EBV activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), a highly potent and selective antiherpes agent, which was recently shown to inhibit several parameters of EBV infection in vitro. When administered intraperitoneally at 200 mg/kg/day for 4 weeks, or 500 mg/kg/day for 2 weeks, starting immediately after B-95-8 cell inoculation, BVDU effectively reduced tumor growth and VCA expression of either subcutaneously or intracerebrally inoculated B-95-8 cells.

Human Lymphoma-Lymphoma Hybrids and Lymphoma-Leukemia Hybrids. II. Epstein-Barr Virus Induction Patterns<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

In addition to the spontaneous expression of markers of the early and late phases of the viral cycle [Epstein-Barr virus (EBV), early viral cycle antigens (EA), and viral capsid antigen (VCA)] by a Panel of hybrid cell lines derived by fusion of human hematopoietic cell lines, the induction of these markers by three chemical inducers [5-iodo-2'-deoxyuridine, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), and sodium butyrate] were analyzed. A variant of the prototype producer of lytic EBV, P3HR-1 (PICAT) was observed to show consistent low spontaneous and induced production of EA and VCA as compared to the P3HR-1 line from which it was derived. An "autohybrid" (PICATPO-1) between these two lines showed a low production of VCA. Hybrids between Raji and P3HR-1 sublines (RUD-PICAT-1 and RUDPUT-2) showed reduced expression of EBV antigens. Both hybrids were capable of VCA expression, in contrast to the Raji parent that expressed EA but not VCA. A new Raji-Daudi hybrid (DITRUD-1) expressed spontaneous and induced EA and VCA at levels intermediate between its two parents. A different type of hybrid was derived from a Daudi subline and the K562 leukemia cell line (DUTKO-1) and was found to be capable of spontaneous as well as induced EA and VCA expression. Both DUTKO-1 and DITRUD-1 were similar to Daudi in their induction profile in respect to a very low response to TPA.

Effect of Adenine Arabinoside on Epstein-Barr Virus in Vitro

The effect of adenine arabinoside (ara-A) on Epstein-Barr virus (EBV) in cultures of lymphoid cells was examined with use of an EBV-producing cell line, P3HR-1, and a nonproducing cell line, Raji. The presence of EBV-associated viral capsid antigen (VCA) and early antigen (EA) was detected by indirect immunofluorescence. Ara-A inhibited the expression of VCA in P3HR-1 cells at concentrations fivefold below those that inhibited cell multiplication; there was no concomitant accumulation of EA. Ara-A did not inhibit superinfection of Raji cells with EBV and did not induce EA until high concentrations of the compound were reached. The inhibition of the expression of VCA but not EA is consistent with a postulated inhibition by ara-A of viral-directed synthesis of DNA. At low concentrations, ara-A may exert a more specific antiviral effect than that reported for idoxuridine and cytosine arabinoside in this system.