Electron microscope study of the base sequence homology between simian virus 40 and human papovavirus BK.

The base sequence homology between the genomes of simian virus 40 (SV40) and human papovavirus BK (BKV) was studied by the heteroduplex method of Ferguson and Davis (J. Mol. Biol. 94:135-149, 1975). When mounted for microscopy in 30% formamide (Tm-35 degrees C), BKV/SV40 heteroduplexes were an average of 92% double-stranded and contained only two small nonhomologous regions that mapped near the junctions between the early and late regions of the SV40 Genome. At higher formamide concentrations, the fraction of duplex DNA in the BKV/SV40 heteroduplexes decreased, indicating significant base mismatching in the homologous regions. The strongest regions of homology were located in the late region.     

Regulatory DNA sequence conserved in the course of BK virus evolution

Summary Within the genome of human polyomavirus BK (BKV), there exists a noncoding regulatory region toward the late region side of the origin of DNA replication. In most BKV strains isolated by viral culture, this regulatory region contains tandem repeats varying in size. Recently. however, several laboratories isolated new BKV strains (designated as archetypal strains) lacking such repeat sequences. To examine the genetic relationship between archetypal strains, a phylogenetic tree was constructed for seven BKV strains, including three archetypal strains, from DNA sequence data on the late genes, those for leader protein (agnoprotein), and those for structural proteins (VP1, VP2, and VP3). For three strains data previously reported were used, whereas for the others sequences were determined in this study. From total numbers of nucleotide substitutions in each pair of strains, a phylogenetic tree was constructed by the unweighted pair-group method. The phylogenetic tree obtained reveals that BKV strains containing the archetypal regulatory region do not constitute a cluster of closely related strains and that these strains, together with those carrying the major part of the archetypal regulatory region, are widespread in the BKV population. This finding suggests that the basic structure of the archetypal regulatory region has been conserved in the course of BKV evolution.     

BK virus T antigens induce kidney carcinomas and thymoproliferative disorders in transgenic mice.

Renal adenocarcinomas and/or extremely enlarged thymuses (up to 250 times normal size) were observed in 60 of 78 mice in a transgenic line containing a single copy of the BK virus (BKV) early region. Enlarged thymuses from different mice displayed thymoproliferative disorders of varying severity, ranging from extreme hyperplasia to thymomas and lymphomas. All kidney tumor DNAs analyzed contained highly amplified BKV sequences with multiple rearrangements in cellular DNA flanking the transgene, whereas amplification and rearrangement were observed only in some enlarged thymus DNAs. Expression of BKV T antigens was restricted to epithelial cells of kidney tumors and enlarged thymuses and was not detected in any normal tissues. Although thymocytes proliferated to numbers much greater than normal in the enlarged thymuses, no T antigen expression was detected in thymocytes.     

Comparison of JC and BK human papovaviruses with simian virus 40: DNA homology studies.

Studies were performed to ascertain the relationship of human papovavirus JC to BK virus and to simian virus 40 (SV40) by further restriction endonuclease analysis and by DNA-DNA competition hybridization on membrane filters. Form I DNA extracted from two new isolates from cases of progressive multifocal leukoencephalopathy of human papovaviruses that were JC-like in their antigenic properties were found to yield restriction endonuclease fragmentation patterns similar to those of prototypic JC virus DNA and different from those of BK or SV40. Form I DNA preparations of JC and BK viruses were found to be related to each other and to SV40 DNA to a similar extent, with JC and BK virus DNAs containing sequences homologous to both early and late regions of the SV40 genome. The relatedness in each comparison was less than 50%, and heterologous hybrids between either JC or BK and SV40 DNAs were found to be less stable than homologous SV40-SV40 hybrids in high concentrations of formamide, suggesting substantial mismatch within homologous regions, to the extent of 15 to 30%. The new JC-like isolates were also studied in competition hybridization reactions with SV40 DNA and yielded results similar to those obtained with JC virus.     

Regions of the polyoma genome coding for T antigens.

The early region of the polyoma genome encodes three T antigens. We have analyzed the organization of the coding regions for the T antigens, using the nucleotide sequence of polyoma DNA and peptides derived from purified, radio-labeled T antigens, separated by two-dimensional electrophoresis and chromatography. We compared the peptides, predicted from the nucleotide sequence of the DNA, with those derived from the purified T antigens. We also compared chemically synthesized peptides, predicted from the DNA sequence, with observed peptides. The results show that the three polyoma T antigens are encoded in overlapping regions of the viral DNA, translated, in part, in two different reading frames.     

Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication.

Base substitution of the ori region of simian virus 40 leads to plaque morphology mutants with markedly decreased DNA replication. Second-site mutations within the simian virus 40 T antigen gene suppress the plaque phenotype and replication defect of base-substituted ori mutants. Two second-site mutations have been mapped to a small segment of the T antigen gene, just beyond the distal splice junction. DNA sequence analysis revealed a single missense change in this segment of the T antigen gene of each of these second-site revertants, leading to a change in codon 157 in one case and codon 166 in the other. The mutant T antigens displayed relaxed specificity for the ori signal, i.e., they can function with several variously modified ori sequences, including those with small nucleotide deletions or insertions that are inactive for replication when coupled with wild-type T antigen. Thus a region of T antigen has been identified that appears to be intimately involved in vivo in binding to the ori sequence to initiate viral DNA replication.     

BK virus-transformed inbred hamster brain cells. I. Status of the viral DNA and the association of BK virus early antigens with purified plasma membranes

Inbred LSH hamster brain cells were transformed in vitro by the GS strain of BK virus (BKV), and transplantable tumors classified as undifferentiated glioblastomas were induced in the syngeneic host. The viral status in the transformed cells, designated LSH-BR-BK, was established. About 46 genome equivalents per cell of viral DNA was detected, with the majority of sequences in a free form. The transformed cells expressed large quantities of tumor (T) antigen as well as surface (S) antigen as demonstrated by indirect immunofluorescence. Sixty-three percent of tumor-bearing hamsters produced high-titer antibodies against T, whereas 3 of 14 (21%) hamsters also produced antibodies against the BKV-specific S antigen. Furthermore, the relatedness of BKV early gene products, including T, S, and tumor-specific transplantation antigen, was established by the production of a rabbit antiserum against highly purified plasma membranes of LSH-BR-BK cells and by the induction of a BKV-specific tumor-specific transplantation antigen response by these plasma membranes in the syngeneic host.     

Functional role of BK virus tumor antigens in transformation.

We have examined the role of the human papovavirus BK virus (BKV) tumor (T) antigen(s) in the maintenance of transformation and have identified the domain of T antigen essential for transformation. BKV-transformed BHK 21 and NIH 3T3 cells expressing antisense T-antigen RNA lose their ability to grow in soft agar, indicating the need for the continued expression of T antigen for the maintenance of the transformed phenotype. Experiments using translation termination linker insertion and deletion mutagenesis of BKV T antigen demonstrate that amino acids 356 to 384 are essential for transformation. Although BKV T antigen shares 100, 95, and 82% amino acid homology with that of simian virus 40 (SV40) for the nuclear localization signal, p53-binding domain, and DNA-binding domain, respectively, the transformation domains of BKV and SV40 T antigens share only 54% homology. Also, BKV T antigen lacks a substantial portion of the ATPase domain of SV40, and our results indicate the dispensability of the remaining portion for transformation by this protein. We suggest that the differences in the amino acids in the identified transformation domains together with the differences in the ATPase domains may account for the differences in the transformation potentials of the two proteins.     

Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens.

The monomer form of BK virus (BKV) tumor antigen (T Ag) was immunoprecipitated from extracts of BKV-transformed cells and had a molecular weight of approximately 113,000. This compared with 97,000 for the molecular weight of either BKV or simian virus 40 (SV40) T Ag from lytically infected cells. The SV40 and BKV T Ag's from productively infected cells were compared by examining their methionine-labeled tryptic peptides. Out of a total of 20 SV40-and 21 BKV-specific peptides, there were seven pairs of similar peptides on the basis of ion-exchange chromatography, These coeluting peptides contained approximately 25 to 30% of the total methionine radioactivity. Similar results were obtained when the tryptic peptides of SV40 T Ag from lytically infected cells were compared with those of BKV T Ag from virally transformed cells.     

The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens.

Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.     

An evaluation of automated homology modelling methods at low target template sequence similarity

MOTIVATION: There are two main areas of difficulty in homology modelling that are particularly important when sequence identity between target and template falls below 50%: sequence alignment and loop building. These problems become magnified with automatic modelling processes, as there is no human input to correct mistakes. As such we have benchmarked several stand-alone strategies that could be implemented in a workflow for automated high-throughput homology modelling. These include three new sequence-structure alignment programs: 3D-Coffee, Staccato and SAlign, plus five homology modelling programs and their respective loop building methods: Builder, Nest, Modeller, SegMod/ENCAD and Swiss-Model. The SABmark database provided 123 targets with at least five templates from the same SCOP family and sequence identities 相似文献   

DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species.

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.