Down-regulation of integrated Epstein-Barr virus nuclear antigen 1 and 2 genes in a Burkitt lymphoma cell line after somatic cell fusion with autologous EBV-immortalized lymphoblastoid cells.

Epstein Barr virus (EBV) latent gene expression was analyzed in somatic cell hybrids between an EBV-positive Burkitt lymphoma (BL) cell line (BL 60) and an autologous EBV-immortalized lymphoblastoid cell line (LCL, IARC 277). The EBV genomes carried by the parental cell lines differ in sequence and in their physical state. The BL 60 EBV genome is integrated into the host cell genome whereas the LCL IARC 277 carries exclusively episomal EBV molecules. The hybrid cells contain both EBV genomes and display the differentiation phenotype of the parental LCL with regard to growth characteristics and cell-surface antigen expression in vitro and in vivo. While the EBNA 1 and EBNA 2 gene expression of the LCL-derived EBV is maintained in these hybrid cells, the BL-60-derived EBNA 1 and EBNA 2 genes are transcriptionally down-regulated. Mapping of the genomic region surrounding the latent Cp promoter of the BL-60-derived EBV revealed an extensive deletion upstream of the Cp promoter including the enhancer element in the ori P region, the origin of latent viral replication (ori P), the coding sequences for the EBV latent membrane protein (LMP) and the EBV terminal protein (TP), and suggested that one viral-cellular junction sequence is located near the Cp promoter. Integration of EBV into the host cell genome together with the extensive deletion might be causally related with the altered latent gene expression pattern after introduction of a lymphoblastoid host-cell background by somatic cell fusion. Down-regulation of the BL-60-derived EBNA genes could be due to loss of regulatory sequences in the BL-derived EBV necessary for EBNA 1 and EBNA 2 transcription in the lymphoblastoid hybrid cells, but not in the parental BL cells.

Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers.

Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.

Subclinical Epstein-Barr virus infection of both the male and female genital tract--indication for sexual transmission.

Epstein-Barr Virus (EBV) can infect B lymphocytes as well as epithelial cells of the oral cavity. Recently, infection of epithelial cells of the inflamed uterine cervix has been demonstrated, and EBV-DNA has been detected in urethral discharge of men suffering from genital infection. We investigated whether EBV can be found in the genital tract of both sexes independently from inflammatory disease states. Genital specimens of men and women of a sexually transmitted diseases outpatient clinic after excluding sexually transmitted diseases and clinically apparent signs of inflammation were investigated using the polymerase chain reaction to screen for EBV-DNA. In 13 of 47 samples (27.7%) swabbed from the uterine cervix, EBV-DNA could be detected. Similarly, 6 of 45 samples (13.3%) scraped from the sulcus coronarius contained EBV-DNA. Our study shows that the female genital tract and likewise the male genital tract can subclinically harbor EBV. These findings suggest i) that in addition to the oral cavity, the female and the male genital tract may be a reservoir for EBV and ii) that sexual transmission of this virus associated with an epidemiology different from that of oral infection may be possible.

Detection of Epstein-Barr virus-DNA in tongue epithelium of human immunodeficiency virus-infected patients.

Oral hairy leukoplakia is a lesion on the lateral part of the tongue that contains replicating Epstein-Barr virus (EBV) and presages progression from human immunodeficiency virus (HIV) infection to AIDS. To clarify the role of EBV in the development of the lesions, we used filter in situ DNA hybridization to determine the prevalence of EBV and of human papillomavirus (HPV) in epithelial cells obtained on swabs from the tongue of HIV-infected patients who had hairy leukoplakia, HIV-infected patients who did not have hairy leukoplakia, and healthy uninfected control persons. In samples collected from the 35 uninfected control persons, EBV DNA could not be detected except at low concentrations in three people. In contrast, all but one of the samples from 11 HIV-infected patients who had hairy leukoplakia contained EBV DNA. Of greatest interest, in 19 of 32 HIV-infected patients who had no signs of hairy leukoplakia, EBV DNA was also detected on the epithelium of the tongue. DNA filter in situ hybridization for the detection of HPV serotypes 6, 11, 16, and 18 in all cases yielded negative results. Statistical analysis showed that the presence of EBV DNA was significantly correlated with the clinical status of the HIV-infected persons, as determined by Walter Reed staging classification, whereas hairy leukoplakia was not. It is concluded that detection of EBV DNA in oral epithelium may be an earlier and more powerful predictor of progression to AIDS than is hairy leukoplakia.

Analysis of the HSV-1 strain 17 DNA polymerase gene reveals the expression of four different classes of pol transcripts.

We have investigated the structure and the expression of transcripts of the HSV-1 strain 17 DNA polymerase gene (pol) by various mapping methods including cDNA cloning. The majority of mature pol transcripts is strictly colinear with the pol gene. But additionally, pol cDNAs show a defined heterogeneity in respect to their 5'-terminal regions and can be divided into four classes with characteristic differences; (i) class 1 represents the major transcript (pol-R1) with initiation at HSV-1 positions 62,605-62,610, (ii) class 2 initiates about 70 bp downstream, (iii) class 3 is generated by splicing the short open reading frame (SORF) to a 5'-truncated part of the long open reading frame (LORF) which results in a partially different coding potential, and (iv) class 4 starts 120 bp upstream of the major initiation site in the central part of the origin of replication (oriL). S1 and Exo VII nuclease and RNase protection assays as well as primer extension analyses confirm the classification regarding the genuine structure of pol mRNAs and the differential usage of transcriptional start sites. Furthermore, the transcript classes can be distinguished from each other by their kinetics of appearance/disappearance in the cytoplasm: The first transcription of the pol gene is indicated by the predominant presence of class 2 and class 4 mRNAs at 2 hr postinfection (h.p.i.), followed by an increase of class 1 transcripts up to 4 h.p.i. and a parallel decrease of class 2 mRNAs. These data suggest that expression of the pol gene is finely regulated already at the transcriptional and/or posttranscriptional level prior to the translation of pol mRNAs.

Diagnosis of Epstein-Barr virus infection in hairy leukoplakia by using nucleic acid hybridization and noninvasive techniques.

The presence of Epstein-Barr virus (EBV) DNA in the epithelial cells of oral hairy leukoplakia is the confirming criterion in the diagnosis of this lesion, which occurs mainly in persons infected by the human immunodeficiency virus. Because hairy leukoplakia often presages the development of the acquired immune deficiency syndrome, it is important that suspicious lesions be accurately diagnosed. Commonly, biopsy tissue is removed for detection of EBV DNA by in situ hybridization, but biopsy is contraindicated in some patients. This study evaluated filter and cytospin in situ hybridization, two noninvasive techniques that examine epithelial cells swabbed from the surfaces of the lesions, for their sensitivity in detecting EBV DNA. As compared with tissue in situ hybridization, the filter and cytospin techniques had sensitivities of 100 and 92%, respectively. We conclude that these two noninvasive techniques can provide the clinician with an accurate alternative to biopsy whenever this human immunodeficiency virus-associated lesion is suspected.

[EBV replication and therapy of oral hairy leukoplakia using acyclovir]. / EBV-Replikation und Therapie der oralen Haarleukoplakie mit Acyclovir.

The therapy of choice for oral hairy leucoplakia in HIV-infected patients is treatment with acyclovir. During treatment the replication of Epstein-Barr Virus (EBV) in cells scraped from the epithelium of hairy leucoplakia was investigated using filter-in-situ hybridization. On the 2nd day of treatment a slight and on the 5th day a marked reduction of the replication was observed, and on the 8th day of treatment replication of EBV could hardly be detected. At that time a marked regression of leucoplakia was seen. Within another 7 days the lesions had completely disappeared. These findings demonstrate the relationship between treatment with acyclovir and inhibition of EBV replication in hairy leucoplakia and the relationship between inhibition of EBV replication and remission of hairy leucoplakia.

TPA-inducible Epstein-Barr virus genes in Raji cells and their regulation.

A number of agents including the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can induce an abortive virus cycle in the EBV nonproducer Burkitt's lymphoma line Raji. We describe the pattern of viral RNAs transcribed in uninduced cells and in cells treated with TPA for 8 hr, as analyzed by Northern blotting. By comparing the patterns of RNAs observed in cells treated with TPA, TPA plus cycloheximide, or cycloheximide alone, we have tested whether any EBV gene in TPA-treated Raji cells would be inducible directly by TPA in the presence of protein synthesis inhibitors, similarly to immediate-early genes induced by superinfection of Raji cells with P3HR-1 virus in the presence of cycloheximide. We demonstrate here that induction of all early EBV genes is dependent on ongoing protein synthesis. The experiments do not provide an answer to whether TPA acts by activating an initial step in the cascade of virus production or whether TPA has a simultaneous pleiotropic effect on the regulation of a large number of viral genes.

Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene.

The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSV-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at Tm - 25 degrees C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Epstein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein.

Geographical prevalence of two types of Epstein-Barr virus.

The Jijoye EBV strain is characterized by a substitution of 1.8 kb in the C-terminal part of the EBNA 2 gene compared to B95-8 or M-ABA virus. This made it possible to construct hybridization probes specific for M-ABA (type A) and Jijoye viruses (type B), which have been used to type the EBV genomes in 38 spontaneously established cell lines. Type A is more prevalent being found in 31 of 38 cases; type B virus was found in five cell lines (Jijoye, LY 67, QIMR-GOR, BL 16, and BL 29); and two cell lines, Daudi and EB-3, contained neither the M-ABA- nor the Jijoye-specific sequences. EBV type B appears to be less ubiquitous, since all type B isolates, including AG 876 virus, originated from Central Africa, La Réunion, and New Guinea. All the other cell lines, carrying EBV type A, were established from patients from Central Africa (4), North Africa (7), New Guinea (1), and Asia (6) and from white individuals (13). The restricted geographical localization of EBV type B in parts of the southern hemisphere and its similarity to herpesvirus papio (T. Dambaugh, K. Hennessy, L. Chamnankit, and E. Kieff (1984) Proc. Natl. Acad. Sci. USA 81, 7632-7636) could suggest that such viruses may have evolved by recombination of EBV with a related Old World monkey virus, alternatively, evolution of virus variants within the human species also being conceivable.

Structure and evolution of two related transcription units of Epstein-Barr virus carrying small tandem repeats.

Two regions of the Epstein-Barr virus (EBV) genome carrying partially homologous clusters of short tandem repeats (NotI and PstI repeats) flanked by 1044 and 1045 base pairs with almost complete homology (DL and DR, left and right duplication, respectively) were most abundantly transcribed into poly(A)+ mRNA after induction with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The nucleotide sequence of both repeat clusters and the conserved upstream regulatory sequences from the M-ABA EBV strain are presented. Nearly the whole part of the sequences coding for the RNAs is covered by the NotI and PstI repeats, respectively. The regulatory sequences for these genes are located in the homologous regions of 1044 and 1045 base pairs (DL and DR, respectively). A CAAT box, a TATA box, and other herpes simplex virus-like elements were identified for both transcription units. The initiation points and the 3' ends of both inducible RNAs were mapped by S1 nuclease analysis. Both genes have open reading frames and may potentially code for proteins with repetitive amino acid compositions. The structure of these two inducible EBV genes is discussed, and an evolutionary model is proposed for the generation of gene duplication in the M-ABA strain of EBV.

Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion.

We conducted a study to identify the viruses in tissue specimens of oral "hairy" leukoplakia, a lesion that is found in immunosuppressed male homosexuals and that is associated with the subsequent development of the acquired immunodeficiency syndrome. When stained for papillomavirus core antigen, 49 of 67 biopsy specimens (73 per cent) yielded positive results in epithelial-cell nuclei. Electron microscopy showed papillomavirus-like particles in all of 25 specimens, and the herpes-type virus described in a previous report was seen in 23 of the 25 specimens. Three specimens had both types of particle in the same individual epithelial cells. Immunofluorescence for herpes simplex virus, varicella-zoster virus, and cytomegalovirus gave negative results in all cases, but 19 of 20 specimens showed intense nuclear staining in epithelial cells for the viral capsid antigen of Epstein-Barr virus (EBV). DNA hybridization using EBV probes in Southern blots demonstrated EBV DNA in all of 13 specimens and found 200 or more viral DNA molecules per cellular genome in 11 of the 13. The whole EBV genome was also demonstrated in the specimens and found to be in linear virion form. We conclude that EBV replicates within the epithelial cells in hairy leukoplakia.

A putative transforming gene of Jijoye virus differs from that of Epstein-Barr virus prototypes.

The P3HR-1 strain of Epstein-Barr virus (EBV), a nontransforming clonal derivative of Jijoye (EBV), is characterized by a deletion of 6.6 kb involving part of the BamHI-W repeats and the adjacent region including the NotI repeats. In the transforming parental Jijoye virus this region differs from the corresponding regions in B95-8 or M-ABA virus. The HindIII-B fragments which carry this region from both Jijoye and prototype M-ABA (EBV) viruses have been cloned and subclones have been constructed which contain the left-hand part of HindIII-B from the HindIII to the BglII site (BglII-delta C fragment). By restriction enzyme analysis the inserts were found to be of equal size (6.3 kb) but to differ in their restriction enzyme pattern. Heteroduplexes formed under stringent conditions in the presence of T4 gene 32 protein revealed a substitution loop of 1750 +/- 200 nucleotides. Heteroduplex formation under nonstringent conditions showed that the substituted sequences are partially homologous to each other, with the regions of nonhomology confined to three distinct areas of 100 to 200 nucleotides. The partial homology observed between both regions indicates that they have evolved from a common ancestor. By hybridization of a Jijoye virus subclone containing only sequences of the substituted region to Northern blots a 2.8-kb polyadenylated transcript was detected indicating that the substituted region is expressed in Jijoye cells.

Structure and transcription of human papillomavirus sequences in cervical carcinoma cells.

DNA of human papillomavirus (HPV) types 16 and 18 has been found closely associated with human genital cancer, supporting the concept that members of this virus group are key factors in the aetiology of genital cancer. HPV 18 DNA sequences were also detected in cell lines derived from cervical cancer. We have now analysed these cell lines, HeLa, C4-1 and 756, for the structural organization and transcription of the HPV 18 genome and we find that the HPV 18 DNA is integrated into the cellular genome and is amplified in HeLa and 756 cells. Almost the complete HPV 18 genome seems to be present in 756 cells, with the early region being disrupted into two portions in each integrated copy. In HeLa and C4-1 cells, a 2-3 kilobase (kb) segment of HPV 18-specific sequences is missing from the E2 to L2 region. HPV 18 sequences are specifically transcribed from the E6-E7-E1 region into poly(A)+ RNAs of 1.5-6.5 kb. Hybridization analysis of cDNA clones indicated that some of the transcripts are composed of HPV 18 and cellular sequences. In addition, poly(A)+ RNA hybridizing with HPV 16 DNA was found in two out of three cervical carcinoma biopsies.

c-myc and immunoglobulin kappa light chain constant genes are on the 8q+ chromosome of three Burkitt lymphoma lines with t(2;8) translocations.

We have determined the localization of c-myc and the immunoglobulin kappa light chain genes on the 8q+/2p- chromosomes of the three Burkitt lymphoma lines BL21, LY66 and LY91 with t(2;8) translocation by in situ hybridization. BL21 is characterized by a complex translocation in which a piece of chromosome 9 appears to be located between the fragments of chromosome 8 and 2 on the 8q+ chromosome. Our data indicate that in all three cell lines the c-myc gene is located on the 8q+ chromosome proximal to the breakpoint in band 8q24. In all cell lines examined the cluster of kappa variable genes has remained on the 2p- chromosome. In LY91 cells the major part of the joining region remained on 2p-, while the joining region has moved to 8q+ in the cell lines BL21 and LY66. In all three cell lines the constant kappa light chain gene was found on the 8q+ chromosome. The fact that an essentially identical pattern was found in the cell line BL21, with the complex translocation, suggests that the insertion of the piece of chromosome 9 into the 8q+ chromosome might be a secondary event. Our present data fit into the concept that in all Burkitt lymphoma lines investigated so far, including cases with t(8;14) and the variant translocations t(2;8) and t(8;22), the c-myc gene becomes situated at the 5' side of an immunoglobulin constant gene. This may have implications for the generation of somatic mutations in the coding and non-coding part of the c-myc gene.

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates.

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

Two distant clusters of partially homologous small repeats of Epstein-Barr virus are transcribed upon induction of an abortive or lytic cycle of the virus.

The two regions of the Epstein-Barr virus genome carrying partially homologous clusters of short tandem repeats (DSL and DSR [duplicated sequences, left and right, respectively] ) are transcribed into polyadenylated RNA upon spontaneous or chemical induction of the lytic virus cycle. In Raji, an Epstein-Barr virus genome carrying a nonproducer cell line, transcription of DSL and DSR is only observed upon induction of an abortive life cycle of the virus. In the nonproducer cell line Raji, the polyadenylated transcripts of DSL and DSR are about 2,500 and 2,700 bases, respectively, in length. Four different spontaneous Epstein-Barr virus producer lines, M-ABA, CC34-5, QIMR-WIL, and B95-8, differ in the length of their DSL and/or DSR regions by different numbers of tandem repeats. The size of the RNAs corresponds in all cases to the size of the respective cluster of repeats, indicating that a large part of each RNA species is colinearly transcribed from the entire tandem repeat arrays. Both the DSL and the DSR RNAs have the same polarity proceeding from right to left on the Epstein-Barr virus genome. DNA sequence analysis of the DSR repeat revealed that translation of the RNA would be possible in three open reading frames within the repeat cluster. Short homologies to herpes simplex virus IR-TR sequences and to immunoglobulin switch region sequences (IgH-S) are discussed.