RESUMEN
The primary nucleotide sequence of three regions of BK virus (BKV) variant (MM) DNA has been determined. The region between map positions 0.715 and 0.900 includes the initiation points and partial coding sequences of the putative VP2 and VP3 proteins of BKV(MM), the amino acid sequences of which show over 80% homology with those of VP2 and VP3 of simian virus 40. The sequence of a potential leader protein X, 66 amino acids long for BKV(MM) and 62 long for simian virus 40, is also deduced. The regions between 0.595 and 0.398 and 0.310 and 0.175 include the coding sequence for the entire small t antigen and most of the large T antigen of BKV(MM). The DNA sequence within these regions comprises over 50% of the complete BKV(MM) genome and shows a 70% sequence homology with the corresponding regions of simian virus 40 DNA. This high degree of homology is at variance with the reported homology values of 11--20% estimated by hybridization measurements of heteroduplex analyses. Possible explanations for the discrepancy are discussed.
Asunto(s)
Virus BK/análisis , ADN Viral , Poliomavirus/análisis , Virus 40 de los Simios/análisis , Secuencia de Bases , Código Genético , Hibridación de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The nucleotide sequence of the late region of the polyoma virus genome has been deduced, which codes for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. The amino acid sequence of VP1 predicted from the nucleotide sequence is in good agreement with the partial N-terminal sequence 1 and amino acid composition of VP1 2,3. When both nucleotide and amono acid sequences are compared with their counterparts in the related viruses, SV40 4,5 and BKV (R. Young, personal communication), extensive homologies are found along the entire regions of the viral genes. Maximum homologies appear to occur in the regions which code for the C-terminal of VP1, on the contrary of the result of heteroduplex analysis 6 with 6 with SV40 and polyoma virus DNAs.
Asunto(s)
Virus BK/análisis , ADN Viral , Genes Virales , Poliomavirus/análisis , Virus 40 de los Simios/análisis , Secuencia de Bases , ADN Viral/metabolismo , Código GenéticoRESUMEN
A total of 51 restriction sites were recognized within the BK virus genome by the combination of 10 different restriction endonucleases. These sites were mapped and oriented relative to one another as well as to the five fragments generated by the digestion of BK virus DNA with HindIII and EcoRI. The result was a comprehensive physical map suitable for in-depth characterization of the functions of BK virus at the molecular level.
Asunto(s)
Virus BK/análisis , Enzimas de Restricción del ADN/metabolismo , ADN Viral/análisis , Poliomavirus/análisis , Virus BK/genética , Genes ViralesRESUMEN
A new restriction endonuclease, SacI from Streptomyces achromogenes cleaves BK virus (strain MM) DNA into 3 fragments, whereas MboII from Moraxella bovis and AluI from Arthrobacter luteus give 22 and 30 fragments, respectively. All these specific DNA fragments were ordered and mapped on the viral genome by two methods first, by the reciprocal digestion method using uniformly 32P-labeled DNA; and second, by the partial digestion technique using the single-end 32P-labeled DNA. This study, together with those reported earlier, defined the location of 90 cleavage sites on the BK virus DNA.
Asunto(s)
Virus BK/análisis , ADN Viral/análisis , Genes Virales , Poliomavirus/análisis , Virus BK/genética , Enzimas de Restricción del ADN , ADN Viral/metabolismoRESUMEN
Most humans in the United States have been infected with BK virus (BKV), a human papovavirus. Because BKV has oncogenic properties, we have investigated whether it may be a cause of human cancer. Basic principles of tumor virology imply that BKV-induced tumors should contain BKV DNA sequences. Therefore, we assayed (by molecular hybridization) DNA from human tumors and malignant cell lines for BKV DNA, using BKV [(32)P]DNA as probe. The BKV [(32)P]DNA was labeled in vitro (nick translation) to specific activities of 1 to 2 x 10(8) cpm/mug. The BKV DNA used to prepare our probes had the properties expected of authentic BKV genomes, including density of superhelical DNA, sedimentation velocity in alkaline and neutral sucrose gradients, production of one fragment by endonuclease EcoRI cleavage and four fragments by endonuclease Hin II + III cleavage and reassociation properties. From these studies we conclude that our BKV probes hybridized well, and represented bona fide BKV DNA. Using three different BKV [(32)P]DNA probes, i.e., from three distinct plaque isolates, we have analyzed DNA from BKV-transformed cells, normal human tissues, and a large number of human tumors. All human DNAs (cell lines, normal tissues, tumors) hybridized 5% with BKV DNA. Hybridization analysis of BKV-transformed hamster cell DNA indicated 5-6 copies of at least 88% of the BKV genome per cell. No BKV DNA sequences were detected (above the normal 5% hybridization to all human DNAs) in the following normal human tissues: 10 kidney (BKV is usually isolated from urine), 3 spleen, 13 lung, 23 colon, 2 rectum, 1 ileum, and 1 skin. No BKV-specific DNA was found in 166 tumors, including 5 carcinomas (Ca) of stomach, 3 Ca small intestine, 26 Ca colon, 9 Ca rectum, 31 Ca lung, 9 adenocarcinomas and 5 oat cell carcinomas of lung, 17 melanomas, 5 Ca prostate, 4 Ca bladder, 6 Wilms tumors, 4 hypernephromas, 15 Ca kidney, 7 brain tumors, 5 Hodgkin lymphomas, 10 lymphomas (immunosuppressed patients have a high incidence of lymphomas), 2 reticulum cell sarcomas (spleen), and 3 skin tumors. We have also analyzed 7 human malignant cell lines (melanoma, lung, rhabdomyosarcoma, and glioblastomas), including several clones of a lung melanoma line; no BKV DNA sequences were detected. Because our probes could detect one copy of BKV DNA if only 10% of the cells were tumor cells, our results are very strong evidence that the tumors we analyzed did not have a BKV etiology. The tumors we tested represent about 50% of all cancers in the United States; there is no evidence that BKV is involved in the etiology of these types of tumors.
Asunto(s)
Virus BK/análisis , ADN Viral/análisis , Neoplasias/análisis , Poliomavirus/análisis , Línea Celular , Transformación Celular Viral , Humanos , Hibridación de Ácido NucleicoRESUMEN
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.
Asunto(s)
Virus BK/análisis , ADN Viral/análisis , Conformación de Ácido Nucleico , Poliomavirus/análisis , Virus 40 de los Simios/análisis , Secuencia de Bases , Microscopía Electrónica , TemperaturaRESUMEN
The DNA of three cloned lines of hamster kidney cells transformed by human papovavirus BK DNA was examined by reassociation kinetics for viral sequences and found to contain 2.7 to 5.3 equivalents of viral DNA per diploid genome. In the one line examined with the four R-HindIII fragments of the human papovavirus BK genome, the entire viral genome was uniformly represented.
Asunto(s)
Virus BK/análisis , Transformación Celular Neoplásica , ADN de Neoplasias/análisis , ADN Viral/análisis , Poliomavirus/análisis , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Células Clonales , Cricetinae , Enzimas de Restricción del ADN/metabolismoRESUMEN
A number of hybridization techniques have been used to assess the homology between the genomes of BK virus (BKV) and simian virus 40 (SV40). A noncontiguous set of homologous sequences has been localized primarily within the late region of the SV40 genome, and these sequences presumably account for the cross-reaction between V-antigens of the two viruses. The reason for the relatively strong crossreaction between SV40 and BKV T-antigens is still unclear. The sequence homology and similarity in genomic organization suggest a close relationship between these papovaviruses.
Asunto(s)
Virus BK/análisis , ADN Viral , Genes , Papillomaviridae/análisis , Polyomaviridae , Poliomavirus/análisis , Virus 40 de los Simios/análisis , Secuencia de Bases , Enzimas de Restricción del ADN , Desoxirribonucleótidos/análisis , Haemophilus influenzae/enzimología , Microscopía Electrónica , Hibridación de Ácido Nucleico , Especificidad de la EspecieRESUMEN
We have determined the DNA sequences which correspond to the splicing regions in the transcripts of human papovavirus BKV, an evolutionary variant of SV40. To precisely localize the excision points in the BKV sequence, we have conducted a preliminary analysis of numerous viral and eukaryotic splice site sequences. This analysis suggests that the preferred sequence for the donor site belongs to at least one of four groups: Pu↓GTAxG, Pu↓GTAxxT, Pu↓GTxxGT, Pu↓GTxAG (↓ = the cleavage site). These four groups derive from the two basic sequences, Pu↓GTxxG and Pu↓GTA. An optimal donor site might be: AG↓GTAAGT. The preferred sequence for the acceptor site is of the form PyPyxPyAG↓; no dinucleotide AG occurs within 13 nucleotides prior to the terminal AG of the 3' end of the intervening sequences. As we have found in this study of BKV splice sites, the sequences for the preferred donor and acceptor sites provide predictive value in localizing RNA splice points when the DNA sequence is known.
Asunto(s)
Virus BK/análisis , ADN Viral , Poliomavirus/análisis , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Viral/metabolismo , Genes Virales , Humanos , Virus 40 de los Simios/análisis , Especificidad de la Especie , Transcripción GenéticaRESUMEN
Extensive physical mapping revealed that approximately 90% of the genomes of BKV(prototype, WT) and BKV (MM strain) are identical or closely related. Nucleotide sequences of the non-homologous regions and a large portion of the homologous regions have been determined for both genomes. The coding sequence of small t antigen of BKV(MM) is 216 nucleotides shorter than that of BKV(WT), even though no differences in biological function of the t antigen was observed. Both genomes contain three similar sets of 44-61 base-pair repeated sequences. However, the DNA sequence of the tandem repeats is totally different between BKV (human cell as host) and SV40 (monkey cell as host). On the other hand, the region between the N-terminus of the T antigen genes and the origin of replication is dominated by a similar set of palindromic sequences in BKV and SV40 DNA. There is also extensive homology between the regions which code for proteins in BKV and SV40, suggesting a close evolutionary relationship.
Asunto(s)
Virus BK/análisis , ADN Viral , Papillomaviridae/análisis , Polyomaviridae , Poliomavirus/análisis , Virus 40 de los Simios/análisis , Antígenos Virales , Composición de Base , Secuencia de Bases , Enzimas de Restricción del ADN , Genes Virales , Biosíntesis de Proteínas , Especificidad de la Especie , Transcripción GenéticaRESUMEN
Primary human urinary cells support growth and plaque formation by papovavirus BK, permitting plaque purification of the virus. After plaquing, the virus forms uniform plaques and its DNA has a homogeneous restriction enzyme fragment profile. Support of BK replication as well as morphological and cultural characteristics suggest an epithelial origin for the cells.
Asunto(s)
Virus BK/crecimiento & desarrollo , Poliomavirus/crecimiento & desarrollo , Orina/citología , Virus BK/análisis , Virus BK/aislamiento & purificación , ADN Viral/análisis , Humanos , Orina/microbiología , Ensayo de Placa Viral , Cultivo de VirusRESUMEN
DNA and RNA sequencing techniques were used to obtain the sequence surrounding the origin of DNA replication for human papovavirus BKV. The structure is characterized by a true palindrome of 17 residues followed by two sets of symmetrical sequences and a stretch of 20 AT residues. Within the two symmetrical sequences is a segment containing a strong purine bias, 23 of 26 nucleotides. These structures are similar, if not identical, to those found in the region of the SV40 replication, origin. Within the homologous DNA segments, 60-80% of the BKV and SV40 nucleotides are the same. The remarkable similarity of BKV and SV40 sequences containing the origins of DNA replication would appear to confirm our previous suggestion of an evolutionary relationship between the two genomes. In addition, topological similarities between these sequences suggest the possibility of certain structural requirements for bidirectional replication origins in these superhelical DNAs.
Asunto(s)
Virus BK/análisis , Replicación del ADN , ADN Viral/análisis , Poliomavirus/análisis , Virus 40 de los Simios/análisis , Replicación Viral , Secuencia de Bases , Enzimas de Restricción del ADN , ARN Viral/análisisRESUMEN
Specific cleavage of BK virus (MM) DNA with restriction endonuclease MboI gives rise to 10 fragments. Two techniques were used to determine the location of these fragments on the viral genome with respect to the three known sites for HindIII cleavage. In the first method, reciprocal digestion, individual MboI fragments were digested with HindIII and individual HindIII fragments were digested with MboI. In the second method, single-end 32P-labeled HindIII subfragments were partially digested with MboI, and then the sizes of the radioactive partial products were used to deduce the nearest neighboring fragment. Information from these two methods is more than adequate to map all the MboI enzyme sites. Cleavage of BK virus (MM) DNA with restriction enzyme HaeIII produces 21 fragments. With the aid of the same two methods, these fragments have also been ordered with respect to the known map locations of the HindIII and MboI sites.
Asunto(s)
Virus BK/genética , Enzimas de Restricción del ADN/metabolismo , ADN Circular/genética , ADN Viral/genética , Genes Virales , Poliomavirus/genética , Virus BK/análisis , Mapeo Cromosómico , ADN Circular/análisis , ADN Viral/análisisRESUMEN
DNA sequences from BK virus have been detected by measurements of reassociation kinetics in DNA preparations isolated from five of 12 human tumor tissues and three of four human tumor cell lines; no DNA sequences from BK virus could be found using the same assay in DNA samples obtained from nontumor tissues removed from various patients. Whether or not this association is trivial, indicative of infection by BK virus, or correlative with the tumor state must await the testing of additional samples of normal and tumor tissues as well as a definition of the physical state of the genome of BK virus.
Asunto(s)
Virus BK/análisis , ADN de Neoplasias/análisis , ADN Viral/análisis , Poliomavirus/análisis , Antígenos Virales/análisis , Línea Celular , Humanos , Terapia de Inmunosupresión , Neoplasias/tratamiento farmacológico , Virus 40 de los Simios/inmunologíaRESUMEN
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.
Asunto(s)
Virus BK/análisis , ADN Viral/análisis , Papillomaviridae/análisis , Polyomaviridae , Poliomavirus/análisis , Virus 40 de los Simios/análisis , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Formamidas/farmacología , Humanos , Leucoencefalopatía Multifocal Progresiva/microbiología , Conformación de Ácido Nucleico , Hibridación de Ácido NucleicoRESUMEN
Human papovavirus, RFV, isolated from urine of a renal transplant patient was compared with two strains of SV40 and with the prototype human papovavirus, BKV. Neutralization tests showed that RFV and BKV are indistinguishable, while large-plaque (LP) and small-plaque(SP) isolates of SV40 gave a low but significant level of cross-reaction with rabbit or human antisera against RFV. DNA reassociation saturation tests using 125I-labelled RFV DNA show that BKV has 88% homology, and SP-SV40 has 29% homology to RFV. We conclude that RFV and BKV are nearly, if not totally, identical and are not SV40 variants.
Asunto(s)
Virus BK , Papillomaviridae , Polyomaviridae , Poliomavirus , Virus 40 de los Simios , Antígenos Virales/análisis , Virus BK/análisis , Virus BK/inmunología , ADN Viral/análisis , Epítopos , Humanos , Trasplante de Riñón , Conformación de Ácido Nucleico , Papillomaviridae/análisis , Papillomaviridae/inmunología , Papillomaviridae/aislamiento & purificación , Poliomavirus/inmunología , Virus 40 de los Simios/análisis , Virus 40 de los Simios/inmunología , Orina/microbiologíaRESUMEN
Four isolates of human papovaviruses (RF, GS, DW, and MG viruses) obtained from the urine specimens of renal allograft recipients in widely separated locations were compared with BK virus. Hemagglutination inhibition tests and plaque neutralization assays showed that all were antigenically related to BK virus. All isolates transformed baby hamster kidney cells, transformation being determined by the ability of the cells to plate in soft agar. Purified preparations of each isolate were iodinated with chloramine T and the polypeptide compositions were compared by electrophoresis of disrupted viruses in polyacrylamide gels containing sodium dodecyl sulfate. The gel patterns of all isolates were similar to that of BK virus. The tryptic digests of two major iodinated virion proteins, VP1 and VP3, were analyzed on an ion exchange column. The peptide patterns of GS, DW, and BK virus were identical; those of RF and MG virus closely resembled the patterns of the above three with only minor differences in some peptides. The results show that the four isolates and BK virus are antigenically closely related, have similar onocogenic potential, and are distinguishable from simian virus 40 and JC virus.
Asunto(s)
Papillomaviridae , Polyomaviridae , Orina/microbiología , Antígenos Virales/análisis , Virus BK/análisis , Virus BK/inmunología , Línea Celular , Epítopos , Humanos , Trasplante de Riñón , Papillomaviridae/análisis , Papillomaviridae/inmunología , Papillomaviridae/aislamiento & purificación , Péptidos/análisis , Virus 40 de los Simios/inmunología , Proteínas Virales/análisisRESUMEN
Two new human papovavirus isolates (JMV and MMV) from the urines of patients with Wiskott-Aldrich syndrome were morphologically and serologically identical to BK virus (BKV). The genomes of these two new isolates were found to be indistinguishable from prototype BKV DNA in a variety of nucleic acid hybridization experiments. Like BKV DNA, JMV and MMV DNAs share approximately 20% of their polynucleotide sequences with simian virus 40 DNA. The genome of JMV was indistinguishable from that of BKV by restriction endonuclease analysis; MMV DNA contained three instead of four R-Hind cleavage sites and one rather than no R-HpaII cleavage sites. Physical maps of the BKV and MMV genomes were constructed using restriction endonucleases, and these maps were oriented to the map of simian virus 40 DNA.
Asunto(s)
Virus BK , Genes , Poliomavirus , Virus BK/análisis , Secuencia de Bases , Mapeo Cromosómico , Enzimas de Restricción del ADN/metabolismo , ADN Circular/análisis , ADN Viral/análisis , ADN Viral/metabolismo , Humanos , Peso Molecular , Conformación de Ácido Nucleico , Polinucleótidos/análisis , Virus 40 de los Simios/análisis , Orina/microbiología , Síndrome de Wiskott-Aldrich/microbiologíaRESUMEN
DNA was prepared directly from preparations pf papovavirus excreted in the urine of 2 patients on immunosuppressive therapy, without preliminary amplification in cell cultures. EcoRI, BamHI, and HindIII restriction patterns were typical of BK virus DNA and were identical for the two isolates. However, there were significant differences in the sizes of the HindIII fragments when compared with those of standard cell-culture-adapted BK virus strains.
Asunto(s)
Virus BK/análisis , ADN Viral/análisis , Poliomavirus/análisis , Animales , Virus BK/aislamiento & purificación , Células Cultivadas , Cricetinae , Enzimas de Restricción del ADN , Desoxirribonucleasa BamHI , Desoxirribonucleasa EcoRI , Desoxirribonucleasa HindIII , Fibroblastos , Humanos , Orina/microbiologíaRESUMEN
Evidence has accumulated indicating that BK virus, following an inapparent primary infection, persists in the renal organs of normal healthy individuals and reactivates upon immunosuppression. Data to support this hypothesis are presented and suggest that BK virus DNA sequences are present at very low levels in the kidneys of more than 50% of the population and that this persistence is localized in several foci within these organs.