The Epstein-Barr virus encoded cytokine viral interleukin-10 enhances transformation of human B lymphocytes.

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in their growth transformation and establishment of immortalised lymphoblastoid cell lines. The virus was recently found to encode a homologue of the pleitropic cytokine interleukin-10 (IL-10), which has wide ranging effects on the immune system. We have investigated the effect of this virally encoded growth factor on the ability of EBV to immortalize B lymphocytes from tonsils and from adult and neonatal blood. Recombinant viral interleukin-10 (vIL-10) was found to increase dramatically the growth transformation of B cells from all three populations infected with either the highly transforming type 1 strain B95-8 or the less efficient type 2 strain BL16. This striking enhancement of transforming ability in the presence of viral IL-10 may be in part due to increased viability of the B cells during infection and decreased levels of interferon-gamma, a cytokine known to inhibit EBV transformation. Thus viral IL-10 influences a number of cell types of the immune system to allow the enhanced outgrowth of EBV transformed cells.

Scanning the structure and antigenicity of HPV-16 E6 and E7 oncoproteins using antipeptide antibodies.

The structure and antigenicity of the HPV-16 E6 and E7 oncoproteins was studied using a set of antisera against overlapping synthetic peptides. We report that antigenic, mobile regions of the native proteins, as defined by reactivity with antipeptide antisera, occur at the N-termini of both E6 and E7 proteins, corresponding to regions of known or suspected protein-protein interactions. The putative zinc finger domains were consistently non-reactive, despite computer predictions of relatively high antigenicity, suggesting that the proposed zinc finger regions are held in stable secondary structures that the peptides were not able to mimic. In E6, the linker region between the two zinc fingers was antigenic, indicating that the two zinc finger structures might be able to articulate relative to one another by a flexible linker region. The highly antigenic N-terminal region of HPV-16 E7 was also found to be antigenic in E7 of both HPV-11 and HPV-18, indicating that the E7 proteins of different HPV types have similar antigenic structures. The identification of antigenic regions of the E6 and E7 proteins should be therefore be useful in the design of site-directed antibodies against E6 and E7 for numerous HPV types.

Epstein-Barr virus gene expression in interferon-treated cells. Implications for the regulation of protein synthesis and the antiviral state.

This paper presents data on the effects of interferon treatment on Epstein-Barr virus (EBV) gene expression in latently infected Daudi Burkitt's lymphoma cells, and reviews the possible role of viral gene products in the regulation of translation. In Daudi cells the main virally coded RNAs are the small untranslated RNAs EBER-1 and EBER-2, two mRNAs for the DNA binding protein EBNA-1, and a number of small RNAs containing sequences from the BamHI W repeat region of the viral genome. Interferon treatment does not change the qualitative pattern of EBV gene expression but decreases the levels of the EBNA-1 mRNAs. The chromatographic behaviour of EBV-encoded RNAs on CF11-cellulose indicates that many contain double-stranded regions; these RNAs co-purify with RNA that activates the interferon-induced, dsRNA-sensitive protein kinase DAI. Computer analysis indicates that the exons transcribed from the BamHI W repeats have the potential for formation of very stable secondary structures. Many viruses can counteract the inhibition of protein synthesis mediated by the DAI-catalysed phosphorylation of initiation factor eIF-2 and our data suggest that the small RNA EBER-1 may fulfil this function in the EBV system. During the infection and immortalization of B lymphocytes by EBV the synthesis of large amounts of EBER-1 RNA might thus allow the virus to circumvent one of the interferon-mediated mechanisms of host cell defence.

Recombinant adeno-associated virus-mediated expression of O6-alkylguanine-DNA-alkyltransferase protects human epithelial and hematopoietic cells against chloroethylating agent toxicity.

Recombinant adeno-associated virus (rAAV) encoding the human O6-alkylguanine-DNA-alkyltransferase (hAT) protein and a selectable marker (Neo(r)) was used to transduce human cervical carcinoma (HeLa) cells and erythroleukemic (K562) cells and clones were selected using G418 (0.4 mg/ml). Thirteen HeLa clones were isolated, 9 of which survived for 2-3 months before cell death ensued, presumably owing to the loss of G418 resistance. Northern blot analysis of the remaining four clones, using a neo probe, showed high levels of RNA equivalent in size to the bicistronic RNA expected to be produced from this construct. Analysis of hAT activity showed that 2000-5000 fmol/mg protein was expressed relative to untransduced cells (800-900 fmol/mg protein). Cell survival analysis following exposure to the chloroethylating agent mitozolomide revealed that expression of hAT at levels two- to fourfold higher than background conferred significant resistance (p < 0.001) to the toxic effects of this drug. Two days following infection of K562 cells with the rAAV vector, immunoblot analysis showed that hAT protein was being produced. Three K562 clones, isolated using G418 selection, were studied in detail and were shown to express hAT activities of 1500, 1010, and 890 fmol/mg protein, respectively, at 40 days posttransduction (mock-transduced K562 cells contain <2 fmol of hAT/mg protein). As with HeLa cells, Northern blot analysis showed the production of an appropriately sized transcript and immunoblot analysis indicated that hAT protein was being produced. These clones were assayed for cell survival following exposure to mitozolomide. Expression of hAT at levels 800- to 1500-fold higher than background conferred significant resistance (p < 0.001) to the toxic effects of mitozolomide. We have therefore successfully conferred a protective advantage against mitozolomide toxicity to cells by rAAV-mediated hAT expression.

Antigen gene transfer to cultured human dendritic cells using recombinant avipoxvirus vectors.

Advances in understanding the role of dendritic cells (DCs) as the major antigen (Ag)-presenting cell type of the immune system combined with the recent development of methods for the ex vivo expansion of human DCs have opened the possibility for the transfer of tumor Ags to DCs with a view toward tumor immunotherapy. In this study, we examined the feasibility of Ag transfer to cultured human DCs using the host range-restricted avipoxvirus, fowlpoxvirus (FWPV). FWPV was found to infect and express a lacZ marker gene in a number of mammalian cell lines of fibroblastic, epithelial, and hemopoietic lineage origins. LacZ recombinant FWPV (rFWPV) was found subsequently to infect human DCs that had been cultured ex vivo from peripheral blood monocytes. Using rFWPV containing lacZ under the control of a vaccinia virus (VV) early/late promoter (p7.5K) and a 10 plaque-forming units per cell multiplicity of infection, >80% of cells expressed the lacZ marker gene. Quantitative analysis showed that the level of expression continued to rise for 5 days postinfection, at which point the experiments were terminated. Replication-competent recombinant VV (rVV) was also shown to be capable of transferring the marker gene to primary DC cultures. However, neither rFWPV nor rVV were able to express transgenes under the control of late viral promoters, indicating that both rFWPV and rVV infections are arrested at an early stage in human DCs. Infection of CD83 + DCs by rFWPV was confirmed by double-staining cytochemistry. We conclude that host range-restricted FWPV can be used efficiently to transfer Ag genes to human DCs ex vivo and may have a role in the development of tumor immunotherapy protocols.

Expression of the Epstein-Barr virus latent membrane protein in nasopharyngeal carcinoma biopsy specimens.

It has been known for some time that the Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). The tumor cells are known to harbor EBV in a latent state. Latently-infected B cells that have become growth transformed by EBV in vitro express some 10 antigens, two of which (Epstein-Barr nuclear antigen 2 [EBNA2] and the latent membrane protein [LMP]) are associated with cellular transformation. We evaluated the expression of these two EBV antigens in NPC by probing tissue sections with monoclonal antibodies. We found that EBNA2 was not expressed and that LMP was expressed in seven of nine biopsy specimens. It is therefore postulated that either there are subsets of NPC or that LMP may be involved only in certain stages of tumor formation.

Human papillomavirus (HPV) genotypes associated with laryngeal papilloma.

Biopsy specimens from 14 patients treated for laryngeal papillomatosis were tested for the presence of human papillomavirus (HPV) genome by the technique of DNA-DNA hybridisation. According to the age of initial presentation, cases were subdivided into juvenile (less than 16 years) and adult onset (older than 16 years) groups. Histological investigation confirmed that it was impossible to distinguish the groups on this basis. Molecular virology using both dot blot and Southern transfer techniques showed that 10 cases carried the HPV type 6 genome, three cases HPV type 11, and in one case no HPV DNA was detected. All six adult onset cases carried HPV 6 sequences while the juvenile onset group comprised four HPV 6 and three HPV 11 cases. In the juvenile onset group more females were affected; in the adult onset group more males were affected. Two of the patients shown to have HPV type 11 sequences in their biopsy material were the most resistant to treatment. One of the adult onset cases subsequently developed a squamous cell carcinoma of the larynx in which HPV 6 DNA was detected. As far as we know this is first time that HPV-DNA has been confirmed in laryngeal papilloma undergoing malignant change.

Bryostatin 1 induces productive Epstein-Barr virus replication in latently infected cells: implications for use in immunocompromised patients.

Bryostatin 1 is a novel anti-tumor agent currently undergoing clinical trial. We investigated the effect of this drug on B-lymphocyte cell lines that carry the Epstein-Barr virus and found that it induces these latently infected cells into the production of transforming virus particles over a wide range of concentrations. These results may have clinical implications, particularly with regard to the use of the drug in the immunocompromised patient.

Evaluation of multiple biologic parameters in cervical carcinoma: high macrophage infiltration in HPV-associated tumors.

A number of diverse biologic parameters have been assessed prior to treatment in a series of patients with cervical carcinoma. Factors analyzed were HPV DNA presence, MHC class I expression, expression of the oncofetal antigen 5T4, the proportions of macrophages, lymphocytes and granulocytes in cell suspensions prepared from tumors, in vitro colony-forming efficiency (CFE) and a measure of intrinsic radiosensitivity, surviving fraction at 2 Gy. Several associations were found. First, HPV DNA-negative tumors contained a small, but significant, decreased number of tumor infiltrating macrophages compared with HPV DNA-positive tumors. Secondly, patients with HPV-positive tumors were significantly younger than those where no HPV was detected. Thirdly, loss of one or more specific alleles in MHC class I positive tumors resulted in higher numbers of tumor lymphocytes and CFEs. Finally, strong expression of the 5T4 antigen was related to a reduction in the proportion of macrophages in tumor cell suspensions. In addition, for stage I and II patients expression of the MHC class I molecule was associated with improved survival compared with patients with tumors where loss of expression was seen.

Infectivity determinants encoded in a conserved gene block of human herpesvirus-6.

The nucleotide sequence was determined for a 9.3 kb BamHI DNA fragment derived from a cosmid clone (Lorist 6) library of the 160 kb human herpesvirus-6 (HHV-6) strain U1102 genome. Analysis of the sequence showed two different sources for the DNA; 8.0 kb was derived from HHV-6, while 1.3 kb was derived from the right repeat of transposon Tn10, the insertion sequence (IS) element IS10R. The IS element sequence is shown to be derived from the host bacteria of the plasmid. The HHV-6 sequence represents a highly conserved part of the genome encoding 15% of the genes conserved among the other human herpesviruses in only 5% of the genome. Six genes were identified, five encoding products with amino acid sequence similarity to homologues in herpes simplex virus (HSV), Varicella Zoster Virus (VZV), human cytomegalovirus (CMV) and Epstein-Barr Virus (EBV). All had closest amino acid similarity to CMV proteins. Three clustered structural genes, included glycoprotein H, a major conserved determinant of infectivity, were jointed to a putative dUTPase homologue in an arrangement distinct to CMV and HHV-6. In the other herpes viruses these genes are separated by over 50 kb. The gene at this point of genetic rearrangement had no sequence similarity to proteins of other herpesviruses. However, there is a protein at this locus in CMV with similar composition and character. Both appear to be highly glycosylated, secreted glycoproteins with repetitive elements similar to those of human mucins. Comparison of sequence available in the HHV-6 GS strain also shows this to be a variable region (5% nucleotide differences) in an overall conserved DNA sequence (0.5%).

Towards gene therapy of Hurler syndrome.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler's syndrome. However, due to a lack of matched related donors and unacceptable morbidity of matched unrelated transplants, this therapy is not available to all patients. Therefore we have been developing an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. A number of different gene transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of haemopoietic colonies as around 50%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. Indeed a possible disadvantage has been identified for the use of intensive transduction procedures. The enzyme is secreted into the medium and functional localisation has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This pre-clinical work forms the basis for a clinical trial of gene therapy for Hurler syndrome.

[Investigation of EB virus DNA BamHI W fragment, EBNA-2 types and EBNA expression in benign and malignant nasopharyngeal biopsies].

Nasopharyngeal biopsies from 96 cases of clinically suspected nasopharyngeal carcinoma and chronic nasopharyngitis were investigated using EB virus DNA BamHI W fragment and EBNA-2 A, B type probes. There was a significant dominance of EBV EBNA-2 A type infection on all samples: nasopharyngeal carcinoma (NPC) 27/35 (77.1%), clinically suspected NPC 7/10 (70.0%), chronic nasopharyngitis (CNP) 18/27 (66.7%). Both W and A probes positive were 20/29 (69.0%) in NPC and 8/25 (32.0%) in non-NPC, indicating a close relationship between the infection of EBNA-2 A type virus and NPC in this series. Meanwhile, the expression of EBNA on 35 cases of biopsy were detected using the anti-complement immunofluorescence method, 19/20 (95.0%) were positive in the NPC group and 1/15 (6.7%) in the non-NPC group. Based on the positive results in detection of A, W or EBNA expression, there was a significant difference between NPC and non-NPC groups (P < 0.001). It is suggested that EBV infection, especially type A, plays an important role in the etiology and pathogenesis of NPC in the Zhanjiang area.

The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin's Reed-Sternberg-like phenotype.

Although the latent membrane protein-1 (LMP1) of the Epstein-Barr virus (EBV) is believed to be important for the transformation of germinal centre (GC) B cells, the precise contribution of this viral oncogene to lymphoma development is poorly understood. In this study, we used a non-viral vector-based method to express LMP1 in primary human GC B cells. Gene expression profiling revealed that LMP1 induced in GC B cells transcriptional changes characteristic of Hodgkin's lymphoma cell lines. Strikingly, LMP1 down-regulated the expression of B-cell-specific genes including B-cell receptor components such as CD79A, CD79B, CD19, CD20, CD22, and BLNK. LMP1 also induced the expression of ID2, a negative regulator of B-cell differentiation. Our data suggest that in EBV-positive cases, LMP1 is likely to be a major contributor to the altered transcriptional pattern characteristic of Hodgkin/Reed-Sternberg cells, including the loss of B-cell identity.

Characterization of the Epstein-Barr virus-encoded thymidine kinase expressed in heterologous eucaryotic and procaryotic systems.

The establishment of mammalian and procaryotic systems which express the Epstein-Barr virus (EBV) thymidine kinase (TK) has been reported previously (E. Littler, J. Zeuthen, A. A. McBride, E. Trøst-Sørensen, K. L. Powell, J. E. Walsh-Arrand, and J. R. Arrand, EMBO J. 5:1959-1966, 1986). The EBV TK activity expressed in both of these systems was characterized by in vitro assays and found to resemble that of the herpes simplex virus TK both in its broad range of nucleoside and nucleotide utilization and also in its ability to accept antiviral nucleoside analogs as substrates. Further results are presented which suggest that these in vitro systems may prove suitable for studying the potential anti-EBV activity of other candidate antiviral compounds.

Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells.

Cytoplasmic RNA prepared from five lymphoid cell lines and a Burkitt lymphoma biopsy was radioactively labeled in vitro and hybridized to cloned EcoRI restriction endonuclease fragments of B95-8 Epstein-Barr virus DNA. The results confirmed that the most abundant cytoplasmic RNA species in such cells is specified by a small region of the genome defined by the EcoRI J fragment. Detailed mapping experiments precisely localized these transcripts within the sequence of the rightmost one-third of the EcoRI J fragment. DNA sequencing suggested that this region of the Epstein-Barr virus genome is unable to code for protein. The major early transcripts consisted of two non-polyadenylated RNA species, each about 170 nucleotides in length. They were both transcribed off the same strand of the DNA and showed significant sequence homology with each other. The coding sequences of the two small RNAs contained potential intragenic control regions for RNA polymerase III.

In vitro transcription of two Epstein-Barr virus specified small RNA molecules.

Cloned DNA from the EcoRI J fragment of EBV has been used as template for in vitro transcription experiments using cell-free extracts prepared from HeLa or KB cells. Two EBV specific RNAs each about 175 bases in length were synthesised and nuclease 51 mapping experiments determined that these in vitro products corresponded precisely to the in vivo species obtained from Raji cells. These two RNA molecules are transcribed by RNA polymerase III and in common with other pol III-synthesised RNAs the coding sequences contain intragenic control regions. The relative abundance of the two RNAs synthesised in vitro differs from that observed in vivo.

Recombinant vaccinia virus induces neutralising antibodies in rabbits against Epstein-Barr virus membrane antigen gp340.

The Epstein-Barr virus membrane antigen gene gp340 was isolated, inserted into several strains of vaccinia virus and expressed under the control of a vaccinia virus promoter. The EBV-derived protein which was produced by the recombinant vaccinia viruses was heavily glycosylated, readily labelled with threonine, could be detected at the surface of infected cells and had a mol. wt. of approximately 340 kd, all of which are properties of the authentic gp340. Polyclonal rabbit antisera against gp340 and an EBV-neutralising anti-gp340 monoclonal antibody both recognised cells infected with the recombinant vaccinia viruses. Moreover, rabbits vaccinated with one of the recombinants produced antibodies that recognised EBV-containing lymphoblastoid cells and neutralised EBV.