[Epstein-Barr Virus LMP1 oncogene variants in cell lines of different origin].

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.

Functionally significant mutations in the Epstein-Barr virus LMP1 gene and their role in activation of cell signaling pathways.

Latent membrane protein 1 (LMP1) of the Epstein-Barr virus is a constitutively activated analog of the tumor necrosis factor receptor TNF-R1. LMP1 serves as a viral oncogene able to transform human B-lymphocytes and rodent fibroblasts via activation of numerous cellular signal cascades. Two specific motifs within LMP1 are responsible for interaction of this viral protein with the receptor protein beta-TrCP/HOS SCF of the ubiquitin ligase E3 complex, playing an important role in degradation of numerous cellular proteins including NF-kappaB inhibitor IkappaBalpha. In this study, we demonstrate for the first time the importance of point mutations affecting HOS-recognizing motifs of LMP1 for activation of NF-kappaB, AP1, and PI3K/Akt signaling pathways. It has also been shown that rat fibroblast cell lines (Rat-1) expressing different HOS mutants of LMP1 produce different amounts of reactive nitrogen species. Our data confirm the hypothesis that point mutations in the C-terminal region of the LMP1 cytoplasmic domain can influence the transforming potential of the Epstein-Barr virus.

[Mutations of the Epstein-Barr virus LMP1 gene mutations in Russian patients with lymphoid pathology and healthy individuals].

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).

[LMP1 gene variants of Epstein-Barr virus in patients with some lymphoproliferative malignancies].

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.

[Structural and functional features of Epstein-Barr virus LMP-1 gene in patients with anaplastic carcinoma of the nasopharynx in Russia]. / Strukturnye i funktsional'nye osobennosti gena LMP-1 virusa Epshteina-Barr u bol'nykh nedifferentsirovannym rakom nosoglotki v Rossii.

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.

The LMP1 gene isolated from Russian nasopharyngeal carcinoma has no 30-bp deletion.

The Epstein-Barr virus (EBV) is tightly linked to the induction of undifferentiated nasopharyngeal carcinoma (NPC), a tumour endemic in certain areas of southeast Asia. The LMP1 gene encoded by EBV is a classical oncogene due to its ability to transform rodent fibroblasts. LMP1 is absolutely essential for transformation of B cells by the virus and is one of the few EBV genes found to be expressed in NPC. It was originally shown that the LMP1 gene from NPC harbours a deletion of 30 bp in the 3' part of the gene. However, this deletion is also present in the virus spread in healthy people of the areas endemic for NPC and also in other EBV-positive tumours as well as in healthy carriers. We isolated and sequenced the LMP1 gene obtained from tissue of 7 Russian patients with NPC and 1 German patient with an NPC-like tumour of the parotid gland (PG) and compared them with the LMP1 gene isolated from peripheral blood lymphocytes (PBLs) of 6 Russian and 4 German healthy EBV-positive carriers. Neither the Russian NPC cases nor the German NPC-like tumour harboured an LMP1 gene with the 30-bp deletion, while 1 Russian and 2 German carriers contained the LMP1 gene with the 30-bp deletion. In addition, the LMP1 gene isolated from PBLs of the German patient was virtually identical to the gene isolated from the primary tumour. Functional analysis showed no correlation between the presence or absence of the 30-bp deletion and the level of induction of the transcription factors NFkappaB and jun/AP-1 caused by LMP1. These data indicate that the 30-bp deletion is not a factor predisposing for NPC. Comparison of the DNA sequences revealed that the LMP1 genes present in the NPCs most likely represent the "strain" persisting in the general population.

[Preparation of T-lymphoblastoid cell lines from patients with T-cell leukemia and asymptomatic carriers of HTLV-I: analysis of the viral genome status]. / Poluchenie T-limfoblastoidnykh kletochnykh linii ot bol'nogo T-kletochnym leikozom i bessimptomnykh nositelei HTLV-I: analiz statusa virusnogo genoma.

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.

Sero-epidemiologic and phylogenetic studies of HTLV-I infection in 2 countries of the Caspian Sea region.

Phylogenetic analysis of HTLV-I variants persisting among population of Turkmenistan and the Republic of Georgia (situated on both sides of the Caspian Sea, not far from a known HTLV-I cluster in Northern Iran) has been carried out. HTLV-I isolates from the above countries were obtained in the course of seroepidemiological investigations. In all, 1,510 blood-donor samples from Turkmenistan and 47 blood samples from hematological patients from Georgia, were tested with different screening and confirmatory assays, including commercial ones. As a result, 7 infected individuals (3 blood donors and 4 relatives of one blood donor) from Turkmenistan and 3 infected individuals (one ATL patient and 2 of his relatives) from Georgia were found. In addition, 4 HTLV-I isolates from Turkmenistan and one from Georgia were cloned and part of their LTR was sequenced. Phylogenetic analysis of sequenced isolates allowed us to conclude that these isolates belonged to sub-type A, one of 3 sub-types of the "Cosmopolitan" type. We found that most of the Turkmenian isolates formed a tight cluster and shared common nucleotide substitutions with isolates originating from Northern Iran (Mashhad). The data obtained suggest that there is a new endemic focus of HTLV-I infection in the Caspian Sea region. Further investigations are required to confirm this hypothesis.

[Features of adult T-cell leukemia virus reproduction in B-cell lines and it's interaction with the Epstein-Barr herpesvirus]. / Osobennosti reproduktsii virusa T-kletochnogo leikoza vzroslykh v B-kletochnykh liniiakh i ego vzaimodeistviia s gerpesvirusom Epshteina-Barr.

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.

[Serological and molecular-biological study of T-cell leukemia virus in Turkmenistan]. / Serologicheskoe i molekuliarno-biologicheskoe izuchenie virusa T-kletochnogo leikoza (HTLC-1) v Turkmenistane.

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.

[Expression of the Epstein-Barr virus terminal repeat protein 1 in Escherichia coli cells]. / Ekspressiia belka terminal'nogo povtora 1 virusa Epshteina-Barr v kletkakh Escherichia coli.

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.

[Immunoreactivity of synthetic peptides, corresponding to B-cellular epitopes of human type I T-lymphotrophic virus structural proteins]. / Immunoreaktivnost' sinteticheskikh peptidov, sootvetstvuiushchikh B-kletochnym épitopam strukturnykh belkov T-limfotropnogo virusa cheloveka I tipa.

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.

Clinical, morphological and virological features of an HTLV-I-positive case of ATL in a white man from the Caucasus.

An HTLV-I-associated case of adult T-cell leukemia (ATL) was described in a 51-year-old white man, native from Georgia, the former U.S.S.R. Clinical manifestation of the disease (enlarged lymph nodes, bone marrow and peripheral blood changes, CNS-involvement, cutaneous lesions and hypercalcemia) as well as laboratory findings were recognized to be very similar to those frequently observed in ATL patients from endemic regions. Mature T-helper surface phenotype detected on peripheral blood lymphocytes of the patient (OKT3-, OKT4+ and OKT8-) and aggressive course of the disease were also in favour of classical type ATL developed in the patient. The HTLV-I antibody presence in an ATL patient was repeatedly confirmed by serological tests (Abbott HTLV-I EIA and Serodia HTLV-I), immunofluorescence and Western blot assay. The latter revealed the presence of a large spectrum of HTLV-I-specific antibodies (to p19, p24, p26, p28, p32, p36, pr53, gp21, gp46, gp62 and gp68 of HTLV-1). The HTLV-I-specific antibodies have also been detected in serum samples of the patient's wife and son. The presence of HTLV-I provirus in the primary ATL patient's PBL was clearly demonstrated by PCR and Southern blot analysis. This case, with the HTLV-I infections detected in two other family members, suggests that in Europe, HTLV-I-positive cases of ATL can occur in virus-infected local people with much wider distribution than that hitherto supposed.

[Differential expression of recombinant hybrid proteins containing amino- and carboxy-terminal regions of the exterior large glycoprotein (gp46) of the human T-cell leukemia virus (HTLV-I) in Escherichia coli cells]. / Differentsial'naia ékspressiia rekombinantnykh gibridnykh belkov, soderzhashchikh amino- i karboksiterminal'nuiu oblasti naruzhnogo bol'shogo glikoproteida (gp46) virusa T-kletochnogo leikoza cheloveka (HTLV-I) b kletkakh Escherichia coli.

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.

[Variants of the interaction of the human T-cell leukemia virus (HTLV-I) with the B-cells of lymphoblastoid lines]. / Varianty vzaimodeistviia virusa T-kletochnogo leikoza cheloveka (HTLV-I) s B-kletkami limfoblastoidnykh linii.

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.