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E O Major

Publications and source records attributed to E O Major.

At least 91 records · Page 5Linked to original sources
PubMed1988

Unusual DNA structure in the regulatory region of the human papovavirus JC virus.

The human papovavirus JC virus (JCV) was analyzed for the presence of unusual DNA conformations. Recombinant plasmids containing 60% of the JCV prototype Mad-1 strain DNA were constructed and analyzed with both enzymatic and chemical probes. Fine-mapping studies revealed that the most prominent S1 nuclease-sensitive and bromoacetaldehyde-modified sites were located within the TATA boxes of each 98-base-pair tandem repeat. Further studies revealed that the S1 nuclease-sensitive site in the first TATA box (proximal to the origin) was approximately 50-fold stronger than the site in the second TATA box (distal from the origin). Deletion of the first TATA box drastically reduced the extent of bromoacetaldehyde modification in the second TATA box, whereas deletion of the second TATA box had little or no effect on the reactivity at the first TATA box. Hence, the biological and conformational role of the second TATA box remains unclear. No supercoil-induced relaxation was found, and reactions with the probes were not pH dependent. Also, fragments containing this regulatory region did not appear to be bent, although the A+T-rich segment contained a tract of eight consecutive A's. We conclude that the regulatory region of JCV contains non-B, but right-handed, DNA conformations which account for this behavior.

BK Virus↗
PubMed1987

Diagnosis of progressive multifocal leukoencephalopathy by brain biopsy with biotin labeled DNA:DNA in situ hybridization.

DNA:DNA in situ hybridization using a cloned JC virus (JCV) DNA probe labeled with biotin confirmed the presence of JCV DNA in formalin-fixed, paraffin-embedded brain biopsies from four cases of progressive multifocal leukoencephalopathy (PML). Only small pieces of tissue were available in each case. Detection of the JC DNA:DNA hybrids was carried out by affinity cytochemistry. JCV DNA was identified predominantly in the nuclei of interfascicular oligodendrocytes in demyelinated areas of the biopsies. JC virus was isolated from one case, and the diagnosis of PML was substantiated in all cases by electron microscopic identification or immunocytochemical labeling of JC viral antigen. In situ hybridization using a biotin labeled JCV DNA probe is a specific, sensitive and convenient method for confirming the diagnosis of PML in suspected cases evaluated by brain biopsy.

Adult↗
PubMed1987

Owl monkey astrocytoma cells in culture spontaneously produce infectious JC virus which demonstrates altered biological properties.

A tumor cell suspension of an explanted JC virus (JCV)-induced owl monkey glioblastoma was inoculated intracranially into four recipient juvenile owl monkeys. Twenty-eight months following inoculation one owl monkey developed a glioblastoma, which was explanted into tissue culture. DNA from both the tumor tissue and tumor cells in culture hybridized to a JCV DNA probe by Southern analysis, indicating that free, as well as integrated, viral DNA may be present. At the time of the second culture passage, viral JCV DNA was extracted from these cells and cloned into a plasmid vector. Nucleotide sequencing of the regulatory region of the cloned DNA demonstrated homology with the prototype Mad-1 strain of JCV and revealed a 19-base-pair deletion in the second 98-base-pair tandem repeat that eliminated a second TATA box. This deletion is characteristic of the Mad-4 strain of JCV, which is highly neurooncogenic. By the third culture passage, 100% of the cells were T-antigen positive. Approximately one-third of the cells in culture hybridized to a biotinylated JCV DNA probe when in situ hybridization was used, a technique that only detects high-copy-number of replicating viral sequences. By the culture passage 5 and continuing through culture passage 14, viable JC virions could be recovered. The T protein synthesized by this virus, now termed JCV-586, differed from both the Mad-1 and Mad-4 strains in that it formed a stable complex with the cellular p53 protein in the tumor cells. Also, the JCV-586 T protein reacted to several monoclonal antibodies made to the simian virus 40 T protein that were not recognized by either the Mad-1 or Mad-4 strains.

Animals↗
PubMed1986

JC virus T protein during productive infection in human fetal brain and kidney cells.

We have examined the synthesis of the T protein of the human polyomavirus, JCV, during productive infection in primary cultures of human fetal glial and kidney cells. Immunoprecipitation of protein extracts from virus infected cells revealed that the JCV large T protein from both the prototype Mad and HEK adapted strains migrated as a 94-kDa protein in denaturing polyacrylamide gels. Resolution of the JCV T protein in brain cells could best be achieved following alkylation of the immunoprecipitated proteins prior to gel electrophoresis. The small t protein of either strain of JCV, however, could not be detected. In comparative experiments, the large T protein of the simian polyomavirus, SV40, was also identified as a 94-kDa protein in immortalized human fetal glial and kidney cultures. There were also protein complexes between p53 and SV40 T protein in the human glial and kidney cell lines. No evidence for a similar protein complex could be detected in JC virus infected human fetal brain or kidney cells.

Antigens, Polyomavirus Transforming↗
PubMed1986

Progressive multifocal leukoencephalopathy: JC virus detection by in situ hybridization compared with immunohistochemistry.

In four cases of progressive multifocal leukoencephalopathy (PML), we compared biotin-labeled DNA:DNA in situ hybridization with peroxidase immunohistochemistry for the detection of JC virus (JCV). The localization of JCV DNA and JCV capsid protein was compared in formalin-fixed, paraffin-embedded brain tissues. Infected oligodendrocytes showed both JCV DNA and JCV protein. However, bizarre astrocytes demonstrated JCV capsid protein less often than JCV DNA. In situ hybridization with a biotinylated probe was as sensitive and specific as immunohistochemistry for diagnosis on formalin-fixed tissue. The presence of both JCV DNA and viral capsid protein in bizarre astrocytes suggests that these cells are neither truly transformed nor permissively infected, but are distinctively altered by JCV.

Adolescent↗
PubMed1985

Progressive multifocal leukoencephalopathy: investigation of three cases using in situ hybridization with JC virus biotinylated DNA probe.

Using the technique of in situ DNA-to-DNA hybridization, a JC virus biotinylated DNA probe was developed and applied to formalin-fixed, paraffin-embedded, or fixed, frozen sections of brain tissue from three subjects with progressive multifocal leukoencephalopathy (PML). Light microscopy was carried out to correlate the presence of JC virus DNA with the selective infection of oligodendrocytes and astrocytes in PML. Oligodendrocytes (lytically infected) showed the greatest evidence of viral DNA. More astrocytes showing bizarre morphological changes had evidence of viral DNA than did astrocytes that were simply reactive. Viral DNA was not evident in vascular endothelial cells using this technique. Viral DNA replication may be an important initial step which produces the bizarre "transformed" astrocytes of PML. Findings in this study do not support the hypothesis that vascular endothelial replication is important in the pathogenesis of JC virus-induced PML. In situ hybridization with biotinylated JC virus probe may be useful in the diagnosis of PML on brain biopsy specimens.

Adult↗
PubMed1985

Establishment of a line of human fetal glial cells that supports JC virus multiplication.

Primary cultures of human fetal brain cells were transfected with plasmid DNA pMK16, containing an origin-defective mutant of simian virus 40 (SV40). Several weeks after DNA treatment, proliferation of glial cells was evident in the culture, allowing passage of the cells at low split ratios. Initially, only 10% of the cells demonstrated nuclear fluorescence staining using a hamster tumor antibody to the SV40 T protein. By the sixth passage, however, 100% of the cells reacted positively to the same antibody. During these early passages, the cells designated SVG began growing very rapidly and acquired a homogeneous morphology. Cell division required only low serum concentrations, was not contact-inhibited, and remained anchorage dependent. These characteristics of the SVG cells have been stable through 25 passages or approximately equal to 80 cell generations. The SV40 T protein is continuously produced in the cells and can direct the replication of DNA inserts in the pSV2 vector, determined by in situ hybridization using biotin-labeled DNA probes, which contains the SV40 replication origin. More importantly, SVG cells support the multiplication of the human papovavirus JCV at levels comparable to primary cultures of human fetal glial cells, producing infectious virus as early as 1 week after viral adsorption. Their brain-cell derivation has been established as astroglial, based on their reactivity with a monoclonal antibody to glial fibrillary acid protein and lack of activity with an anti-galactocerebroside antibody, which identifies oligodendroglial cells. The SVG cells represent a unique line of continuous rapidly growing human fetal astroglial cells that synthesizes a replication-proficient SV40 T protein. Their susceptibility to JC virus (JCV) infection obviates a host restriction barrier that limited JCV studies to primary cultures of human fetal brain and thus should allow for more detailed molecular studies of human brain cells and JCV that infects them.

Antigens, Polyomavirus Transforming↗
PubMed1984

JC virus-induced owl monkey glioblastoma cells in culture: biological properties associated with the viral early gene product.

JCV induces glioblastomas in owl monkeys 18-24 months or longer following intracranial inoculation (W. London, S. Houff, D. Madden, D. Fuccillo, M. Gravell, W. Wallen, A. Palmer, J. Sever, B. Padgett, D. Walker, G. Zu Rhein, and T. Ohashi, 1978, Science 201, 1246-1248). Cells from one brain tumor, owl monkey 26, were successfully established in culture and analyzed for phenotypic characteristics generally associated with persistence and expression of the papovavirus early region of its genome. Owl monkey 26 cells demonstrated nuclear JCV T protein detected by either SV40 hamster tumor sera or PAb 108, a monoclonal antibody to SV40 T protein. However, the JCV T protein was not detected in a complex with the host cell p53 protein as judged by immunoprecipitation using PAb 122, a monoclonal antibody directed to the mammalian p53 cellular protein. These cells also demonstrated increased plasminogen activator secretion and actin cable disorganization properties not before reported for JCV-induced tumor or transformed cells. Of the primate papovaviruses, JCV is unique in its ability to induce brain tumors in these primates. JCV early gene expression can be shown to persist in brain tumor cells once established in culture and correlates with cell phenotypes typical of papovavirus malignant transformation even though the time between virus inoculation and tumor development is usually several years.

Actins↗
PubMed1984

Human embryonic kidney cells: stable transformation with an origin-defective simian virus 40 DNA and use as hosts for human papovavirus replication.

An origin-defective mutant DNA of simian virus 40 immortalized human embryonic kidney cells, maintaining a T protein which could function for human papovavirus BK DNA replication but not for human papovavirus JC DNA replication. Neither BK virions nor capsid proteins were produced in these cells. This may indicate that the simian virus 40 T protein in human embryonic kidney cells is competent for maintaining transformation and initiating and completing DNA replication for BK but is not competent for switching to late gene functions. Furthermore, it appears that the JC DNA replication origin cannot efficiently use the simian virus 40 T protein for its DNA synthesis, as suggested by its DNA sequence data (R. Frisque, J. Virol. 46:170-176, 1983; T. Miyamura, H. Jikoya, E. Soeda, and K. Yoshiike, J. Virol. 45:73-79, 1983).

Cell Line↗
PubMed1983

Human papovavirus infection of an SV40-transformed human embryonic kidney cell line.

Human embryonic kidney cells were transformed at their third passage with an origin-defective mutant of SV40, 1-11. One clone of these cells, SV1, demonstrated several phenotypes of transformation including high-density growth and anchorage independence. The SV1 cells also were 100% T antigen-positive when tested by immunofluorescence and immunoprecipitation assays. These cells with the SV40 T protein were able to support the persistent replication of BK viral DNA but did not support virus multiplication. The SV1 cells would also support the replication of the Sau 3A A fragment of the BK genome cloned into pBR 322. However, SV1 cells would not support multiplication of JC virus or replication of JC DNA.

Cell Line↗
PubMed1983

JC virus T protein expression in owl monkey tumor cell lines.

Four owl monkey tumor cell lines were tested for the presence of JCV early gene products. Owl 26 was the only cell line in which large T protein could be immunoprecipitated using cross-reactive anti-SV40 hamster tumor sera. Small t protein could not be detected in any of the four cell lines. The monoclonal antibody Cl 122, directed against the host-cell 53K protein, could bind the SV40-T-53K host protein complex in human embryonic kidney cells transformed by SV40. However, the Cl 122 antibody only reacted with a 53K protein in Owl 26 not complexed with the JCV T protein.

Animals↗
PubMed1980

Selection of somatic cell hybrids between BK virus transformed BHK-21 and human embryonic kidney cells to study viral gene expression.

Somatic cell hybrids were constructed between the human papovavirus, BKV, transformed BHK-21 cells and human embryonic kidney cells, the permissive host for BKV using the polyethylene glycol-DMSO cell fusion method. Hybrids were selected to preserve the phenotype of transformation as anchorage independence to allow examination of the synthesis of BKV early gene products, the T proteins. Several hybrid clones which survived the selection procedure demonstrated an enhanced production of the small t or approximately 22 K protein while the viral large T or approximatley 94 K protein was not detectable. It was also determined in this study that BHK-21 cells which are thymidine kinase deficient (TK-) can be stably transformed by BKV and appear to remain enzyme deficient after transformation.

Animals↗
PubMed1979

Inhibition of murine sarcoma virus induced transformation in an adenovirus -- NRK system.

Cell transformation induced by murine sarcoma virus (MSV-M) is significantly inhibited (80--90%) in a clonal line of normal rat kidney (NRK) cells when they are infected with rat cell passaged adenovirus 12 (R-Ad12). No inhibition is seen when R-Ad12 is added simultaneously with, or 1 1/2 or 24 hr after, MSV-M infection, suggesting that inhibition occurs most probably intracellularly. There is also a direct correlation between the extent of focus formation and the concentration of R-Ad12 used. Concentrations of R-Ad12 used to inhibit cell transformation do not affect cell growth. Significant inhibition in foci (90--100%) is also evident in the first two subcultures of R-Ad12 infected NRK cells. The mechanism of the inhibition is not yet known.

Adenoviruses, Human↗
PubMed1978

A variant of adenovirus 12 producing cytoplasmic accumulation of capsid proteins.

A variant of adenovirus type 12 (R-Ad12) has been isolated by infecting a clonal line of normal rat kidney cells (NRK) with wild-type adenovirus 12. Although R-Ad12 failed to produce infectious progeny virions when cycled a second time through NRK cells, it nevertheless retained the ability to code for virus structural proteins that accumulated in the cytoplasm.

Adenoviruses, Human↗