A monoclonal antibody was previously described which precipitated two human herpesvirus 6 early proteins. The experiments reported in this paper were designed to determine the relationship of these two proteins. This was determined by probing enzymatically digested viral DNA with a cloned p41 cDNA construct and with a synthetic p38 oligonucleotide. These probes reacted with genomic fragments of different sizes following enzymatic digestion. In addition, the p38 synthetic probe reacted with uninfected cell DNA. These results indicated that p41 and p38 share an antigenic epitope but are encoded by different genomic fragments.

HHV-7 first isolated in 1990 from a healthy individual, is a ubiquitous agent. The second independent isolation of HHV-7 from a chronic fatigue syndrome patient was reported in 1992. The seroepidemiology of HHV-7 suggested that its prevalence rate in the U.S.A. population is > 85%; however, in Japan a low prevalence rate has been reported. HHV-7 can be more readily isolated from the saliva than HHV-6. The primary infection of HHV-7 appears later in life than HHV-6. No disease has been reported that is etiologically linked to HHV-7. HHV-7 is more closely related to HHV-6 and the human cytomegalovirus than other members of the human herpesvirus family.

Experiments were designed in an attempt to identify T- and B-cell epitopes expressed on the 17-kDa early-antigen-restricted (EA-R) polypeptide of the EBV-induced early antigen complex. Using Berzofsky's algorithm, 3 hypothetical T-cell epitopes on p17 were synthesized and employed in EBV-specific lymphoproliferative assays. Lymphocytes from all EBV-infected donors responded against one of these epitopes (p 17.1) irrespective of their serological status relative to antibodies to EA-R. Both CD4+ and CD8+ T-cell subpopulations from seropositive donors proliferated in the presence of p17.1 in short-term cultures. These experiments therefore identified one T-cell epitope on the 17-kDa polypeptide. In contrast, sera from anti-EA antibody-positive individuals reacted with all 3 synthetic peptides to varying degrees, with p17.1 being the most frequently reactive epitope. When the sera were grouped according to diagnosis, it was noted that 82% of the sera from patients with aggressive lymphomas, whether Africans with Burkitt's lymphoma or North Americans with intermediate-grade large-cell or high-grade B-cell lymphoma, contained antibody reactive with p17.1, while 64% were reactive with p17.2 and 29% with p17.3. In contrast, high anti-EA antibody-positive sera from nasopharyngeal carcinoma patients were relatively less reactive with these synthetic peptides (23% positive with p17.1; 19% with p17.2; and 13% with p 17.3). These results therefore identified 3 B-cell EA-R epitopes which might be potentially useful for clinical or epidemiological studies of EBV-associated lymphoproliferative diseases.

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.

Monoclonal antibodies (MAbs) were developed against immunodominant HHV-6 (GS isolate) late and early proteins. The major late protein was identified as a probable glycoprotein with a molecular weight of approximately 110 kDa (gp 110). Immunoblotting of the early antigen yielded proteins of 41 and 38 kDa (p41/38). The MAb to the early protein reacted with cells infected with 14 different HHV-6 isolates. In contrast, the MAb against the late protein reacted with only 10 of these isolates, indicating that there was strain variation in this glycoprotein. The percentage of antibody-positive sera reactive with gp110 in the ELISA ranged from 56% to 96% among the different serum donor categories. In contrast, only 10-30% of the sera were positive for antibodies to p41/38 with the exception of sera from patients with African Burkitt's lymphoma (ABL) and Hodgkin's disease (HD). These antibody patterns denote the presence of active HHV-6 replication in patients with ABL and HD.

An Epstein-Barr virus protein associated with the restricted component of the early antigen complex was characterized in this study. This protein was of particular interest because of its homology to polyoma middle T antigen and to the product of the bcl-2 oncogene. The results from this study reveal that this protein had a number of unusual properties in comparison with other polypeptides associated with the early antigen complex. For example, the synthesis of this membrane-associated antigen was regulated by the cell cycle, and its expression was compatible with cell survival. Functional studies reveal that immunoprecipitates containing this protein exhibited a threonine-serine-specific protein kinase activity. The results suggest that this protein might function in both the immortalization and replication cycles of this virus.

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.

A 3‐step enzyme‐linked immunosorbent assay (ELISA) was developed for detecting IgA antibodies Co purified Epstein‐Barr virus (EBV) polypeptides. The 3‐step procedure included the use of a mouse anti‐human IgA monoclonal antibody (MAb) to amplify the IgA reaction. The 2 major EBV proteins used in this assay were the 125‐kDa component (gpl25) associated with the viral capsid antigen (VCA) complex and a major glycoprotein (gp 250/200) associated with the membrane antigen (MA) complex. Eighty‐two sera were tested on ELISA plates containing either both of the glycoproteins or each one separately. These included 45 IgA antibody‐positive sera from patients with nasopharyngeal carcinoma {NPC). With these sera, there was a good correlation, both qualitatively and quantitatively, between results with the immuno‐fluorescence (IF) and ELISA procedures. Although most IgA antibody‐positive sera contained antibodies reactive with both gpl2S and gp250/200, a number of sera contained antibodies reactive with one of the glycoproteins but not with both. The data indicated that both of these glycoproteins should be used in assays for detecting IgA antibodies to EBV, to avoid false‐negative results. This assay should be useful for screening large populations for IgA antibodies to EBV and also possibly for monitoring disease course in patients with NPC.

By using monoclonal antibodies to different Epstein-Barr virus (EBV) polypeptides in combination with immunoblotting, we detected antigens associated with EBV replication in extracts from nasopharyngeal carcinoma (NPC) biopsy specimens. Major polypeptides associated with both the diffuse and the restricted components of the early antigen (EA) complex were found in extracts from nine of nine NPC biopsy specimens. Cells from an additional NPC biopsy specimen, passaged repeatedly in nude mice, were found to be positive for the major EA (restricted) polypeptide. This approach revealed that extracts from three of 14 biopsy specimens form other benign and malignant diseases also expressed these viral polypeptides. Therefore, for the first time, these results conclusively demonstrate the presence of EA polypeptides in extracts from NPC biopsy specimens. This finding provides at least a partial explanation for the reported prognostic value of antibodies to this antigen in patients with this disease.

When the latent Epstein-Barr virus (EBV) genome in B95-8 cells is induced into a replicative phase, two abundant early RNAs are transcribed rightward from the EBV BamHI H DNA fragment into BamHI F. Analysis of cDNA clones prepared from the RNA of cells replicating EBV revealed that both RNAs contain the BHRF1 open reading frame. Part of BHRF1, cloned into a prokaryotic fusion protein expression vector, expressed a fusion protein in Escherichia coli and the purified fusion protein was used to generate a monoclonal antibody against BHRF1. This antibody was then employed to characterize the protein encoded by BHRF1 in cells replicating EBV. The monoclonal antibody reacted with a 17-kDa protein component of the restricted early antigen (EA) complex. The distribution of the protein in cells was similar to that noted when sera from patients with African Burkitt's lymphoma were used to stain these cells. The protein was synthesized before the major 47–56 kDa protein associated with the diffuse component of EA in superinfected Raji cells. All human sera containing antibodies to EA as determined by immunofluorescence (IF) reacted with the protein as did some sera determined to be anti-VCA positive and anti-EA negative by IF. The predicted amino acid sequence of the protein has characteristics which suggest that it is a membrane protein. It also has significant homology with both the anchor region of polyoma middle T antigen and with the predicted protein product of the bcl-2 mRNA activated by the 14/18 chromosome translocation characteristic of follicular lymphomas. This latter homology is extensive and colinear, suggesting common evolution and function. However, neither a mRNA which could efficiently translate the BHRF1 protein nor the BHRF1 protein could be detected in latently infected cells. Thus, the bcl-2 predicted protein is similar to an EBV protein synthesized in the early phase of virus infection.

The 85-kDa polypeptide previously shown to be associated with the restricted (R) component of the EBV-induced early antigen (EA) complex was subjected to amino acid sequencing analysis. This was accomplished by cyanogen bromide cleavage and by separation of individual peptides by high-pressure liquid chromatography employing reversed-phase C18 column techniques. Two of the isolated peptides, F11 and F13, were subjected to amino acid sequencing and both were found to have significant homology to the postulated protein encoded by the BamHl O right reading frame 2 (BamHl ORF2) of the B95-8 strain Epstein-Barr virus. Computer analysis revealed significant homology between the amino acid sequence of this polypeptide and ribonucleotide reductase, an enzyme previously mapped to this genomic fragment. Amino acid composition analysis also revealed a similar association. These results indicate that the 85-kDa EA(R) polypeptide is associated with a component of the EBV-induced ribonucleotide reductase.