Epstein-Barr virus (EBV) glycoprotein gp350 expressed on transfected cells resistant to natural killer cell activity serves as a target antigen for EBV-specific antibody-dependent cellular cytotoxicity.

Cell surface-associated viral glycoproteins are thought to play a major role as target antigens in cellular cytotoxicity and antiviral immunosurveillance. One such glycoprotein is the Epstein-Barr virus (EBV)-encoded glycoprotein 350 (gp350), which is expressed on both virion envelope and EBV producer cells and carries the virus attachment protein moiety. Although it is known that some antibodies to gp350 can neutralize the virus, the role of this glycoprotein in EBV-specific cellular cytotoxicity is not yet clear. We describe here a study in which we successfully used a new approach to demonstrate that gp350 is a target antigen for EBV-specific antibody-dependent cellular cytotoxicity (ADCC). Transfection of gp350-negative cells resistant to natural killer (NK) cell activity (i.e., Raji) with a recombinant vector (pZIP-MA) containing the gene encoding the EBV-gp350 and the neomycin resistance gene enabled us to isolate cell clones with a stable and strong expression of gp350 on their surface membranes. ADCC determined by using two clones clearly demonstrated that gp350 is the target of the EBV ADCC. Interestingly, this ADCC was comparable to that obtained against the EBV-superinfected (coated) Raji cell expressing the same percentage of gp350 positivity as the two clones. No cytotoxic activity was detected against either nontransfected (gp350-negative) Raji cells or cells transfected with the vector [pZIP-neo-SV(X)1] lacking the gp350 gene. In addition to demonstrating that gp350 is a target molecule for EBV-specific ADCC, our approach in using NK-resistant transfectants provides a lead for probing the role of cell surface-associated viral antigens in specific cellular killing and immunosurveillance.     

Phosphorylation of the Epstein-Barr virus (EBV) DNA polymerase processivity factor EA-D by the EBV-encoded protein kinase and effects of the L-riboside benzimidazole 1263W94.

A member of the family of L-riboside benzimidazole compounds, 1263W94, was shown recently to inhibit replication of Epstein-Barr virus (EBV) (V. L. Zacny, E. Gershburg, M. G. Davis, K. K. Biron, and J. S. Pagano, J. Virol. 73:7271-7277, 1999). In the present report the effect of 1263W94 on the phosphorylation pattern of the EBV DNA polymerase processivity factor, EA-D, during viral reactivation in latently EBV-infected Akata cells is analyzed. This pattern specifically changes with progression of cytolytic infection. In the presence of 1263W94 the appearance of the hyperphosphorylated form of EA-D is mainly affected. Next, coexpression of the cloned EBV-encoded protein kinase (EBV PK), BGLF4, with EA-D demonstrated the ability of EBV PK to phosphorylate EA-D to its hyperphosphorylated form in transient assays. However, the phosphorylation of EA-D was not directly inhibited by 1263W94 in these coexpression assays. The results indicate that the EBV PK appears to be responsible for the hyperphosphorylation of EA-D, imply that the phosphorylation status of EA-D is important for viral replication, and suggest that 1263W94 acts at a level other than direct inhibition of EA-D phosphorylation by EBV PK.     

Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody.

We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme-linked immunosorbent assay) and 0.8 to 1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

Malignant transformation of Epstein-Barr virus-negative Akata cells by introduction of the BARF1 gene carried by Epstein-Barr virus

Spontaneous loss of the Epstein-Barr virus (EBV) genome in the BL cell line Akata led to loss of tumorigenicity in SCID mice, suggesting an important oncogenic activity of EBV in B cells. We previously showed that introduction of the BARF1 gene into the human B-cell line Louckes induced a malignant transformation in newborn rats (M. X. Wei, J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka, Cancer Res. 54:1843-1848, 1994). Since 1 to 2% of Akata cells expressed lytic antigens and expressed the BARF1 gene, we investigated whether introduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Here we show that BARF1-transfected, EBV-negative Akata cells activated Bcl2 expression and induced tumor formation when they were injected into SCID mice. In addition, when EBV-positive Akata cells expressing a low level of BARF1 protein were injected into SCID mice, the expression of BARF1, as well as several lytic proteins, such as EA-D, ZEBRA, and a 135-kDa DNA binding protein, increased in tumor cells while no latent LMP1 and late gp220-320 expression was observed in tumor cells. These observations suggest that the BARF1 gene may be involved in the conferral of tumorigenicity by EBV.     

Accumulation of Epstein-Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji

As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.     

Down-regulation of proteolytic complexes following EBV activation in BL cells

In Burkitt's lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin-proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the alpha/beta subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6' subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.     

Expression and localization of the Epstein-Barr virus-encoded protein kinase

The protein kinase (PK) encoded by the Epstein-Barr Virus (EBV) BGLF4 gene is the only EBV protein kinase. The expression pattern of EBV PK during the reactivation of the viral lytic cycle and the subcellular localization of the protein were analyzed with a polyclonal antiserum raised against a peptide corresponding to the N terminus of EBV PK. Based on previously published data (E. Gershburg and J. S. Pagano, J. Virol. 76:998-1003, 2002) and the expression pattern described here, we conclude that EBV PK is an early protein that requires viral-DNA replication for maximum expression. By biochemical fractionation, the protein could be detected mainly in the nuclear fraction 4 h after viral reactivation in Akata cells. Nuclear localization could be visualized by indirect immunofluorescence in HeLa cells transiently expressing EBV BGLF4 in the absence of other viral products. Transient expression of 3'-terminal deletion mutants of EBV BGLF4 resulted in cytoplasmic localization, confirming the presence of a nuclear localization site in the C-terminal region of the protein. In contrast to the wild-type EBV PK, all of the mutants were unable to hyperphosphorylate EA-D during coexpression or to phosphorylate ganciclovir, as measured by an in-cell activity assay. Thus, the results demonstrate that the nuclear localization, as well as the kinase activity, of BGFL4 is dependent on an intact C-terminal region.     

Inhibition of Epstein-Barr virus DNA synthesis and late gene expression by phosphonoacetic acid.

Growth of lymphoblastoid cells (B95-8, Raji) is not inhibited by the presence of 0.4 mM phosphonoacetic acid. The synthesis of Epstein-Barr virus (EBV) in the producer line B95-8 is completely inhibited, as shown by the total inhibition of viral capsid antigen synthesis. Early viral antigens are made normally in the presence of phosphonoacetic acid, but EBV DNA synthesis is blocked in cells entering the productive cycle. Nonproducer cells in the population replicate the resident EBV DNA by a mechanism that is resistant to phosphonoacetic acid. These results are consistant with the hypotheses that EBV DNA is replicated by two mechanisms, one in the noninduced cell and a different mechanism in the producer cell, and that prior replication of EBV DNA, probably by the second mode, is a prerequisite for late gene expression.     

Human herpesvirus 7: antigenic properties and prevalence in children and adults.

The recent isolation of human herpesvirus 7 (HHV-7) from activated CD4+ T lymphocytes of a healthy individual raises questions regarding the prevalence of this virus in humans and its immunological relationship to previously characterized human herpesviruses. We report that HHV-7 is a ubiquitous virus which is immunologically distinct from the highly prevalent T-lymphotropic HHV-6. Thus, (i) only two of six monoclonal antibodies to HHV-6 cross-reacted with HHV-7-infected cells, (ii) Western immunoblot analyses of viral proteins revealed different patterns for HHV-6- and HHV-7-infected cells, (iii) tests of sequential serum samples from children revealed seroconversion to HHV-6 without concomitant seroconversion to HHV-7, and (iv) in some instances HHV-7 infection occurred in the presence of high titers of HHV-6 antibodies, suggesting the lack of apparent protection of children seropositive for HHV-6 against subsequent infection with HHV-7. On the basis of the analyses of sera from children and adults it can be concluded that HHV-7 is a prevalent human herpesvirus which, like other human herpesviruses, infects during childhood. The age of infection appears to be somewhat later than the very early age documented for HHV-6.     

Characterization of Epstein–Barr virus reactivation in a modeled spaceflight system

Epstein–Barr virus (EBV) is the causative agent of mononucleosis and is also associated with several malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, among others. EBV reactivates during spaceflight, with EBV shedding in saliva increasing to levels ten times those observed pre‐and post‐flight. Although stress has been shown to increase reactivation of EBV, other factors such as radiation and microgravity have been hypothesized to contribute to reactivation in space. We used a modeled spaceflight environment to evaluate the influence of radiation and microgravity on EBV reactivation. BJAB (EBV‐negative) and Raji (EBV‐positive) cell lines were assessed for viability/apoptosis, viral antigen and reactive oxygen species expression, and DNA damage and repair. EBV‐infected cells did not experience decreased viability and increased apoptosis due to modeled spaceflight, whereas an EBV‐negative cell line did, suggesting that EBV infection provided protection against apoptosis and cell death. Radiation was the major contributor to EBV ZEBRA upregulation. Combining modeled microgravity and radiation increased DNA damage and reactive oxygen species while modeled microgravity alone decreased DNA repair in Raji cells. Additionally, EBV‐infected cells had increased DNA damage compared to EBV‐negative cells. Since EBV‐infected cells do not undergo apoptosis as readily as uninfected cells, it is possible that virus‐infected cells in EBV seropositive individuals may have an increased risk to accumulate DNA damage during spaceflight. More studies are warranted to investigate this possibility. J. Cell. Biochem. 114: 616–624, 2013. © 2012 Wiley Periodicals, Inc.     

Expression of Epstein-Barr viral capsid, complement fixing, and nuclear antigens in stationary and exponential phase cultures

Three continuous lymphoblastoid cell lines, 2 productive of nucleocapsids and 1 nonproductive line, were studied for their content of Epstein-Barr viral (EBV) antigens during transition from stationary to logarithmic phase growth. As a preliminary step, viable cells were separated from degenerating ones in discontinuous gradients of serum albumin. Viral capsid antigens were found in both living and dead cells of the 2 producer lines; however, complement fixing (CF) antigens and nuclear antigen were detected only in viable cell subpopulations. The content of antigen detectable in extracts of viable cells by complement fixation remained constant in replicating and resting cultures; further, all viable cells of the 3 lines demonstrated intranuclear antigen by anticomplement immunofluorescence in all stages of growth. In contrast, the proportion of cells with viral capsid antigen in the producer lines increased 7- to 24-fold following entry of resting populations into the phase of exponential growth.The results suggest that expression of viral capsid antigens is discontinuous and is initiated in response to events in log phase, possibly DNA synthesis or mitosis. Expression of the complement fixing and nuclear antigens in continuous in viable cells. These findings emphasize the intimate relationship of the CF and nuclear antigen to the transformed state and suggest that study of this antigen complex will shed light on the mechanisms of lymphocyte transformation by EBV.     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

Effect of acute Plasmodium falciparum malaria on reactivation and shedding of the eight human herpes viruses

Human herpes viruses (HHVs) are widely distributed pathogens. In immuno-competent individuals their clinical outcomes are generally benign but in immuno-compromised hosts, primary infection or extensive viral reactivation can lead to critical diseases. Plasmodium falciparum malaria profoundly affects the host immune system. In this retrospective study, we evaluated the direct effect of acute P. falciparum infection on reactivation and shedding of all known human herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8). We monitored their presence by real time PCR in plasma and saliva of Ugandan children with malaria at the day of admission to the hospital (day-0) and 14 days later (after treatment), or in children with mild infections unrelated to malaria. For each child screened in this study, at least one type of HHV was detected in the saliva. HHV-7 and HHV-6 were detected in more than 70% of the samples and CMV in approximately half. HSV-1, HSV-2, VZV and HHV-8 were detected at lower frequency. During salivary shedding the highest mean viral load was observed for HSV-1 followed by EBV, HHV-7, HHV-6, CMV and HHV-8. After anti-malarial treatment the salivary HSV-1 levels were profoundly diminished or totally cleared. Similarly, four children with malaria had high levels of circulating EBV at day-0, levels that were cleared after anti-malarial treatment confirming the association between P. falciparum infection and EBV reactivation. This study shows that acute P. falciparum infection can contribute to EBV reactivation in the blood and HSV-1 reactivation in the oral cavity. Taken together our results call for further studies investigating the potential clinical implications of HHVs reactivation in children suffering from malaria.     

Detection of four lymphotropic herpesviruses in Hungarian patients with multiple myeloma and lymphoma

It has been suggested that human herpesvirus 8 (HHV-8), also known as KSHV (Kaposi's sarcoma-associated human herpesvirus), might possess a promoting effect in the development and progression of monoclonal gammopathies. In this study, the presence of Epstein-Barr virus (EBV), human cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human herpesvirus 8 (HHV-8) were tested in patients with multiple myeloma (MM) using both serologic and nucleic acid amplification techniques. The transient reactivation or continuous presence of EBV, CMV, HHV-6 and HHV-8 could be detected in, respectively, 36, eight, 13 and 29 of 69 MM patients; nine, one, four and six of 16 monoclonal gammopathy of unknown significance patients; and seven, four, zero and five of 10 Waldenstr?m's macroglobulinemia patients. The total number of MM patients was 95. HHV-8 PCR-positivity was significantly more frequent in the MM group than in the control group of patients with non-Hodgkin's lymphoma (NHL). However, serologic testing did not reveal significant differences between the two patient groups. The number of MM patients with concomitant herpesvirus infections as detected by PCR was as follows: 15 double, seven triple and two quadruple virus nucleic acid positive. In 13/95 MM patients, the simultaneous presence of acute EBV infection and HHV-8 PCR-positivity was detected compared with none of the control group (P=0.009). These results indicate that in addition to HHV-8, the transitional reactivation of EBV may also play a role in the pathogenesis of MM.     

Epstein-Barr Virus-associated Antigens in Non-producing Clones of Human Lymphoblastoid Cell Lines

NUCLEIC acid hybridization suggests that the Epstein-Barr virus (EBV) genome may be present in human lymphoblastoid cell lines that are free of detectable EBV^1,2. We describe here a plentiful appearance of EBV-associated early antigens (EA) and the viral capsid antigen (VCA) in non-producing Raji and NC-37 cell lines when exposed to 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine (IUdR). These antigens were synthesized in all the Raji and NC-37 clones exposed to BUdR or IUdR, strongly suggesting that a complete, but unexpressed, EBV genome exists in the cells of these non-producing lines.