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It is well known that the Epstein-Barr virus (EBV) is a widespread infection in the human population. Typically, infection occurs in early childhood without serious consequences for infected people. At the same time, a secondary infection with an additional EBV strain occurs quite often. During the in vitro cultivation of peripheral blood lymphocytes from persons infected with multiple strains of the virus, only one of these strains with higher transforming potential becomes dominant, while the others are eliminated. Under certain conditions, such a highly transforming EBV strain apparently is able to be the etiologic agent of EBVassociated diseases. To find out the range of highly transforming EBV strains prevalent among Russians, cell lines from patients with EBV-associated and non-associated tumors, as well as healthy individuals, were established. The structural analysis of the latent membrane protein 1 gene (LMP1), a key oncogene of the virus, isolated from established cell lines and peripheral blood lymphocytes of blood donors was carried out, and data obtained were compared with the respective data for LMP1 isolates, amplified from cell lines established from African and Japanese patients with Burkitt's lymphoma. The data obtained show a genetic relationship between Russian LMP1 isolates regardless the fact whether they come from patients with tumors or healthy individuals and differ significantly from LMP1 isolates from Burkitt's lymphoma patients. Thus, the results of the study suggest that in nonendemic region for EBV-associated pathology, Russia, any strain of EBV with any structure of LMP1 with concomitant effect of additional factors may become an etiologic agent for EBV-associated neoplasia.

Epstein-Barr virus (EBV) is an etiological agent of a number of benign and malignant human diseases, such as infectious mononucleosis (IM), Hodgkin's lymphoma (HL), and non-Hodgkin's lymphoma (NHL). EBV latent membrane protein 1 (LMP1) gene (recognized as a viral oncoprotein) of various clinical and geographical origin was found to have different types of amino acid mutations affecting its biological activity. Since there was no information on the strain differences in LMP1 of EBV persisting in Russia, the authors made a sequence analysis of LMP1 samples amplified from the biological materials of Russian patients with IM, HL, and NHL and healthy individuals. The studies have shown that LMP1 variants of Russian origin are a mixed heterogeneous group containing both the earlier characterized and presumably new genetic variants. Among the point amino avid substitutions, the mutations S366T, F106Y, 185L, and E328Q associated with the enhanced transforming activity of a LMP1 molecule and its reduced cytotoxicity. There was no specific association between the certain Russian variants of LMP1 and the specific forms of the disease (IM, HL, and NHL).

The samples of tumor biopsy, blood, and saliva from 10 patients with Hodgkin's disease, 10 patients with non-Hodgkin's lymphoma, and the blood samples of 20 donors were tested by polymerase chain reaction (PCR) for standard (wild) B95-8 and Cao-like (deleted) variants of the LMP1 gene. The paraffin sections of most PCR-tested tumors were also investigated by immunohistochemistry using the monoclonal antibodies S12 or 7D7 to detect the expression of the standard or Cao-like variants of LMP1 protein, respectively. It is suggested that Eptein-Barr virus (EBV) that contains the above deletion is not crucial for the development of the study lymphoproliferative malignancies. The fact that in some cases there is the Cao-like variant of LMP1 in the tumor biopsy specimen and its standard variant LMP1-B95-8 in the biological fluids of the same patient is very likely to suggest that the patient is infected with both types of the virus or there is genetic mutation(s) of EBV during viral carcinogenesis preceding or accompanying the development of a tumor.

The Epstein-Barr virus latent membrane protein 1 (LMP1) is pivotal in the transforming activity of this virus. We found that the common LMP1-95-8 variant interacts with Homologue of Slimb (HOS), a receptor for the SCFHOS/betaTrCP ubiquitin-protein isopeptide ligase (E3) via one canonical and one cryptic HOS recognition site. These sites are mutated or deleted in the tumor-derived LMP1-Cao variant, which did not bind to HOS. Mutations within these sites on LMP1-95-8 abrogated HOS binding and increased transforming activity of LMP1. HOS did not regulate stability of LMP1-95-8 unless it was mutated to bear additional lysine residues near the cryptic motif. LMP1 proteins that could not bind to HOS exhibited an increased ability to induce IkappaB degradation and NF-kappaB-mediated transcription without further increase in activation of IkappaB kinases. Expression of LMP1-95-8 reduced the levels of endogenous HOS available to interact with phosphorylated IkappaBalpha. Degradation of IkappaBalpha and dose dependence of NF-kappaB activation by LMP1-95-8 were promoted by co-expression of HOS. Our data suggest that LMP1-95-8 is a pseudo-substrate of SCFHOS/betaTrCP E3 ubiquitin ligase and that interaction between LMP1 and HOS restricts the extent of LMP1-induced NF-kappaB signaling. We discuss the potential role of this mechanism in transforming and cytostatic effects of LMP1 variants in cells and Epstein-Barr virus-associated tumors.

Epstein-Barr virus (EBV) is known to be closely associated with the development of anaplastic nasopharyngeal carcinoma (NPC) in some malignancy endemic regions in South-East Asia. LMP1 gene is one of the EBV latent genes, which encodes a latent membrane protein. LMP1 gene is thought to be a classical oncogene since it morphologically transforms cells in vitro and induces tumors in experimental animals in vivo. LMP1 is one of a few genes which is expressed in NPC tissues. It was first shown that C-terminus of LMP1 gene obtained from NPC patients in South-East Asia contained a deletion of 30 base pairs (bp). However, this deleted LMP1 gene was then found in the EBV isolates persisting among healthy virus carriers and patients with other EBV-associated abnormalities from both NPC endemic and non-endemic regions. The aim of this investigation was to accomplish a molecular biological analysis of EBV LMP1 genes obtained from Russian NPC patients. To this end, the authors isolated and sequenced the LMP1 clones amplified from the tumor tissues from 7 NPC patients at the N. N. Blokhin Russian Cancer Research Center and primary blood lymphocytes (PBL) from 6 healthy donors. As a result, the authors could not find the deletion of the above-mentioned 30 bp in NPC LMP1 clones, but could in one healthy donor (PBL-2). A functional analysis revealed no significant differences between LMP1 variants with or without 30 bp deletion in their capacity to activate NF kappa B and jun/AP-1 transcription factors. Nevertheless, Russian NPC-derived LMP1 variants as compared with those from PBLs featured some specific amino acid exchanges. These data indicate that the 30 bp deletion of LMP1 gene is not a factor that predisposes to NPC in Russia.

Three HTLV-I-infected partially interleukin-2-dependent lymphoid cell lines were derived from a patient with T-cellular leukemia (ATL) from Georgia and a carrier of HTLV-I from Sakhalin. The strains cultured in the presence of 3-5% interleukin-2 were designated as NBK-1, NBK-2, and YE-1, respectively. Immunoblotting analysis showed typical HTLV-I proteins in them except NBK-2 which expressed nontypical proteins p40K and p28-29K. Unexpectedly, leukemic cell fraction ATL/NBK from a patient contained gag proteins p19 (CA), p24 (MA), Pr53, and unidentified protein p29. Southern blot analysis of primary leukemic cells NBK showed one full-length non-defective provirus with restriction sites Sacl in both LTRs. Limited restriction map of the provirus virtually did not differ from previously described HTLV-I prototypes. Although the mechanism of abnormal protein expression remains to be determined, this event can be explained by defective provirus formation in NBK-2 cell line during coculturing of leukemic cells with human umbilical cord blood lymphocytes.

Interaction between human T-cell leukemia virus (HTLV) with B cells and Epstein-Barr virus (EBV) was studied by immunoblotting, immunofluorescence, virus isolation in permissive T-cell cultures, and polymerase chain reaction (PCR). HTLV-1 in vitro infects the B-cell cultures containing EBV but not EBV-negative cell lines. Productive infection of EBV+ B cells was associated with syncytium formation which led to the elimination of HTLV-1 producing cells. However, the remaining B-cell population contained gag, pol, and pX--the "silent" provirus sequences. HTLV-1 infection of B cells altered the expression of some latent proteins of EBV (EBNA-1, EBNA-2, EBNA-5, and LMP). The changes were represented by increase of molecular weight and/or appearance of additional proteins and were individual for each cell line. Alteration of EBV protein expression may change the functional activity of these proteins, but this hypothesis is to be tested.

Seroepidemiological and molecular-biological screening of 1510 donor blood samples, collected from the residents of the town of Ashgabat (Turkmenistan), for lymphotropic virus of human T-cellular leukemia (HTLV) virus revealed one donor with a high level of immune response to a wide spectrum of viral proteins. Three donors were serologically assessed as dubious, for their sera contained antibodies to gag gene protein but no antibodies to env gene protein. Screening of family members of the donor infected with HTLV-1 revealed four more highly reactive carriers of HTLV-1 virus. The presence of proviral sequences of HTLV-1 in the lymphocyte DNA of infected donor and her relatives was confirmed by polymerase chain reaction and subsequent Southern-blot hybridization of specific amplification products. Proviral sequences of gag, pol, and LTR genes were detected in all the cases. Short-term culturing of peripheral blood lymphocytes of all seropositive subjects was associated with expression of HTLV-1 structural proteins. Analysis of the possible routes of transmission of HTLV-1 isolated in Turkmenistan permits us to hypothesize an Iranian origin of the isolated virus strain.

A pMAL-vector-based plasmid clone with synthetic tac-promotor effectively expressing full-length terminal repeats protein 1 (TP1) of Epstein-Barr virus (EBV) in E. coli was constructed. It is important that the N-terminal region of recombinant TP1 was represented by a maltose-binding protein. The latter can be used to separate TP1 from bacterial lysate by affinity chromatography. Moreover, after treatment with the proteolytic factor Xa, full-length TP1 can be recovered in a discrete form. On the basis of the pATH tryptophan-regulated vector, several plasmid clones expressing different fragments of N- and C-terminal regions of TP1 were also constructed. This collection of recombinant proteins could be used as an important tool for obtaining corresponding antisera and for immunological mapping of the TP1 molecule.

The immunoreactivity of 25 synthetic peptides corresponding to amino acid fragments of the HTLV-I structural proteins p19 gag, gp46 and gp21 env were studied in enzyme linked immunosorbent assay using a serum panel of 70 reference positive specimens with anti-HTLV-I antibodies. The location of the synthetic peptides containing the B-cell epitopes of HTLV-I was established. Anti-HTLV-I antibodies effectively recognized these peptides. The significance of some amino acids for forming the HTLV-I antigenic determinants was estimated. The synthetic peptides with amino acid sequences 100-130 p19 gag and 176-201 gp46 env were found to have most immunoreactivity (90-99% recognition by sera of HTLV-I infected patients) and mimic the immunodominant B-cell epitopes of HTLV-I structural proteins.

Plasmid clones capable of expressing a recombinant fusion proteins containing the anthranilate synthase of E. coli (TrpE) and different regions of gp46 HTLV-I were constructed on the basis of pATH-vectors. A high extent of TrpE-gp46 proteolytic degradation took place independently of the bacterial La-protease. Fusion proteins containing an N-terminal part of gp46 were more stable and could be purified in preparative quantities but were less antigenic. On the contrary, a TrpE-gp46 protein encoded by the TaqI-TaqI DNA fragment and containing only 35 C-terminal amino acids was still susceptible to degradation but possessed good serologic reactivity. Some of the recombinant proteins obtained can be useful for diagnostics and for preparing monoclonal or polyclonal antibodies.

B-cell lymphoblastoid lines which are known to be derived by in vitro inoculation of B-lymphocytes with Epstein-Barr herpes virus (EBV) were shown to be infected with HTLV-1. Three possible variants of HTLV-1 interaction with cells were demonstrated by immunoblot, polymerase chain reaction, and virus isolation: (1) prolonged productive infection; (2) infection of the cells manifested only by the presence of "silent" virus sequences; (3) temporary production of HTLV-1 (3.5 months) after the end of which genetic material persisted in the cells. The long-term productive HTLV-1 infection in EBV-infected B-cells was found to influence the functioning of EBV genome which was manifested by expression of two additional proteins of EBNA-5 group and by changes in the intensity and pattern of LMP and EBNA-2 proteins the functioning of which is associated with immortalizing and transforming properties of EBV.

The results of examinations for the presence of antibody to HTLV-I of 205 sera which were positive or indefinite (doubtful) with regard to HIV are presented. No evidence indicating simultaneous infection with HIV and HTLV-I viruses in 18 HIV-positive subjects was obtained. Antibodies to gag-proteins p19 and p28 HTLV-I were detected in one serum showing primary reactivity in screening for HIV antibodies. Problems of HIV and HTLV-I occurrence and sensitivity of screening test systems are discussed.

The first of case of adult T-cell prolymphocytic lymphosarcoma is reported which according to the natural course, cell composition of tumor, cell surface markers as well as expressed immune response to a wide spectrum of HTLV-1 gag an env encoded proteins can be classified as HTLV-associated adult T-cell leukemia. Diagnosis of the tumor alongside with identification of HTLV-1-infected individuals in the USSR emphasizes the need to stimulate research in HTLV-1-associated carcinogenesis in this country.

The work reported here demonstrates the possibility of rapid generation of highly transforming viruses from rabbit epithelial corneal cells (SIRC) originated from simian sarcoma associated virus (SSAV). The results of the experiments indicate a possible association of the phenomenon of "phenotypic transformation" with the expression of cellular genes with a potential transforming activity. SIRC/SSAV phenotypic transformation was induced by cultivation of these cells in the medium with a low concentration of serum or calcium ions. During subsequent infection of mink lung fibroblasts the authors succeeded in obtaining two new transforming SSAV isolates which induced transformation foci of various morphological types in cellular lines permissive for SSAV replication.

The results of studies of a co-carcinogenic effect of two human infectious viruses in tissue culture are reported here. Viable vaccinia virus actively replicating in the cells of primary BALB/c tissue culture and in a number of continuous murine cell lines has been shown to induce in them expression of major structural p30 protein of murine retroviruses. Vaccinia virus has been also shown to cause biochemical transformation of murine cells. Evidence for the capacity of herpes simplex virus type 2 to induce malignant transformation of BALB/3T3 murine cell line has been obtained and confirmed by transplantation to mice. Transformed cell clones did not contain complete infectious herpes simplex virus but were resistant to superinfection with this virus. N-tropic endogenous murine retrovirus of C type with buoyant density in the saccharose density gradient of 1.18 g/cm3 and a reverse transcriptase activity was expressed in the transformed cells. In the virion structure six proteins typical of these viruses with the prevalence of p30 have been demonstrated. Competitive radioimmunoassay revealed a very high level of virus production: p30 level reached 7500 ng p30 R-MuLV per mg of viral protein. Specificity of this results was shown in control experiments.

The article presents the data obtained by the authors in studies of virus-viral co-cancerogenesis, the interaction between some non-oncogenic viruses and well-known oncogenic viruses, the results of co-cancerogenic effect Marek's disease herpesvirus with the avian leukemia virus and the possibility of phenotype mixing between oncornaviruses belonging to different species in nature.

Possibility of rapid generation of highly transforming viruses from rabbit epithelial corneal cells (Sirc) on the basis of SSAV virus is discussed. The results of the study suggested a relationship between the phenomenon of "phenotypic transformation" with expression of cellular genes and a potential transforming activity. Sirc/SSAV phenotypic transformation was induced by cultivating of cells in a medium with low levels of serum or calcium ions. Subsequent infection of mink lung fibroblasts resulted in generation of 2 new transforming SSAV isolates which induced transformation in cellular lines permissive for SSAV replication. The foci of transformation were of various morphological patterns.