Alignment of the Genome of Monkey B-Lymphotropic Papovavirus to the Genomes of Simian Virus 40 and BK Virus

We located the origin of DNA replication of African green monkey B-lymphotropic papovavirus DNA by analyzing pulse-labeled form I DNA. With the replication origin used as a reference point, the B-lymphotropic papovavirus genome was aligned with the genomes of simian virus 40 and BK virus from DNA homology between specific fragments hybridized under low-stringency conditions. From the results of these experiments, it was possible to deduce the correlation between the physical and functional maps of the B-lymphotropic papovavirus genome.     

Cytoplasmic RNA from normal and malignant human cells shows homology to the DNAs of Epstein-Barr virus and human adenoviruses.

Cytoplasmic RNA prepared from several human cell lines and tissues was hybridised to DNA from Epstein-Barr virus, human adenovirus types 2, 3 and 12 and human papovaviruses BK and JC. RNA from all the cells, regardless of whether or not they were virally infected, hybridised to specific regions of the Epstein-Barr virus or adenovirus genomes but not to papovavirus DNA. The cellular cross-hybridising species appear to be repetitive sequences which are conserved in higher eukaryotes. Mismatch estimations indicate a high degree of homology between the viral and host sequences. Detailed analysis of selected regions of viral DNA failed to reveal any primary-structural peculiarities.     

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).     

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.     

Simian agent 12 is a BK virus-like papovavirus which replicates in monkey cells.

We have begun to characterize the genomic structure and replication of the baboon papovavirus simian agent 12 (SA12). We have defined a wild-type clone of SA12 (SA12 wt100) by plaque purification from a heterogeneous stock. The functional map of SA12 wt100 can be aligned with those of the other primate papovaviruses by assigning one of the two EcoRI sites as 0/1.0 map units. The origin of bidirectional viral DNA replication maps near 0.67 map units, consistent with the limits of sequences homologous to origin sequences in the other papovaviruses. DNA sequence analysis shows that the organization of the SA12 genome is similar to that of the other primate papovaviruses studied. The arrangement and sequence of functional elements in the origin of replication region, as well as the sequences of the N-terminal regions of early protein products, indicate that SA12 is most closely related to the human virus BK, next most closely related to JC virus, and less closely related to simian virus 40. Unlike BK virus, SA12 is capable of productive infection of African green monkey kidney cells.     

Physical map of the BK virus genome.

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.     

Organization of viral genome in a T antigen-negative hamster tumor induced by human papovavirus BK.

We analyzed by blot hybridization the state and structure of the viral DNA in an exceptional BK virus-induced hamster tumor (choroid plexus papilloma Vn-324) that contains about one copy of the BK virus genome per cell, but no intranuclear T antigen as assayed by indirect immunofluorescence. The BK viral DNA was found to be integrated into cellular DNA at a site in the middle of the early region of the viral genome (between 0.32 and 0.41 map units). The structure of the inserted viral DNA shows that it cannot encode a full-size large T antigen, but may encode small T antigen and an N-terminal portion of large T antigen.     

The genome of human papovavirus BKV.

The complete DNA sequence of human papovavirus BKV(Dun), consisting of 5153 nucleotide pairs, is presented. We describe the segments of the genome which correspond to the replication origin, the tandem repeated sequences, the 5' and 3' ends of the mRNAs, the splice sites, the early and late viral proteins and the putative viral polypeptides. These BKV DNA sequences are compared with analogous regions in the SV40 and Py virus genomes in an attempt to localize viral functions for lytic growth and transformation.     

Viable deletion mutant of human papovavirus BK that induces insulinomas in hamsters.

A plaque morphology mutant (pm-522) of human papovavirus BK, which was rescued from a human papovavirus BK-induced hamster pineocytoma, was characterized and compared with a cloned wild-type virus (wt-501). Mutant pm-522 formed turbid plaques and grew more slowly than wt-501 in human embryonic kidney (HEK) cells. The immunofluorescence assay revealed that more HEK cells underwent abortive infection with pm-522 than with wt-501. Whereas wt-501 induced brain tumors and osteosarcomas, but no insulinomas, in hamsters, pm-522 induced brain tumors and insulinomas. The DNA of pm-522 was found by electrophoresis and electron microscopy to have a deletion (85 +/- 15 base pairs) and an insertion (40 +/- 10 base pairs) between map coordinates 0.708 and 0.725 from the endonuclease EcoRI cleavage site. These results demonstrate the presence of a viable deletion human papovarivus BK mutant capable of inducing insulinomas in hamsters.     

Change of DNA near the origin of replication enhances the transforming capacity of human papovavirus BK.

A turbid-plaque-forming mutant (pm522) of human papovavirus BK, which has a small deletion at about 0.7 map unit and grows somewhat more slowly in human cells than does wild-type BK virus, transformed hamster and rat cells in culture much more efficiently than did wild-type virus. Another plaque morphology mutant, pm525, forming turbid plaques larger than those of pm522 also had a high transforming capacity. The similar difference in transforming capability between wild-type and plaque morphology viruses was observed with DNAs extracted from virions. Recombinant viruses were constructed from the wild-type DNA fragment lacking HindIII-C (0.62 to 0.73 map unit) and pm522 HindIII-C (including the origin of replication) by the molecular cloning method. Characterization of the recombinants showed that the change near the origin of DNA replication was responsible both for the altered plaque morphology and for the enhanced transforming capacity of the BK virus mutant.     

Malignant transformation of hamster kidney cells by BK virus.

Primary hamster kidney cells were transformed by BK virus, a new human papovavirus. Transformed (HKBK) cell produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.     

Comparison of JC and BK Human Papovaviruses with Simian Virus 40: Restriction Endonuclease Digestion and Gel Electrophoresis of Resultant Fragments

JC virus was found to have a buoyant density of 1.20 g/cm(3) in linear sucrose-D(2)O and 1.35 g/cm(3) in cesium chloride isopycnic gradients. DNA extracted either from JC-infected cultures or from gradient-purified virions occupied a dense position relative to linear DNA in cesium chloride/ethidium bromide gradients, and the circular configuration of the extracted DNA was confirmed by electron microscopy, with a measured molecular weight of 2.93 x 10(6). DNA from BK virus was similarly prepared and compared to JC and to an SV40 DNA standard by digestion with restriction endonuclease preparations from Haemophilus influenzae, Haemophilus parainfluenzae, and Escherichia coli. Digests were electrophoretically analyzed on gradient polyacrylamide slab gels or agarose gels, and the three viruses were found to have distinctly different cleavage patterns by this form of analysis: JC and BK viruses were almost entirely different from SV40 and significantly different from each other. Thus, JC and BK human papovaviruses appear to be discrete new members of the papovavirus group, rather than SV40 variants.     

Free viral DNA in BK virus-induced hamster tumor cells

The biological properties of nine clonal lines of BK virus-induced hamster tumor cells were studied. All clonal lines were oncogenic and showed an enhanced ability to form colonies in semisolid medium. The cells of each clonal line contained T antigen; no virus could be rescued from any of the clonal lines. The number of viral DNA copies was determined in three of the clonal lines and varied from 10 to 20 copies per diploid amount of cell DNA. The state of the viral genome was studied in these lines, and the great majority of the viral DNA molecules appeared to be present as free (nonintegrated) molecules. At least six length classes of free defective BK virus DNA molecules, which all lacked a part of the late region of the genome, were detected in these cells. Three of the six length classes of BK virus DNA molecules acquired a TaqI recognition site, which suggested substitution of cellular DNA.     

Characterization of human papovavirus BK DNA.

The DNA of the BK virus (BKV) human papovavirus was found to be heterogeneous, consisting of at least four discrete species of DNA. Only the largest of these four species, BKV DNA (i), which has a molecular weight calculated to be 96% that of simian virus 40 (SV40) DNA, was infectious. Homogeneous preparations of BKV DNA were obtained, however, from virions purified after low multiplicity infections of human embryonic kidney cells. BKV DNA (i) was shown to contain a single R-Eco RI and four R-Hind cleavage sites. The R-Eco RI site was localized in the largest R-Hind cleavage fragment. Radiolabeled BKV DNA reassociated slightly faster than SV40 DNA; 20 to 30% polynucleotide sequence homology was demonstrated between the genomes of BKV and SV40 when the reaction was monitored by chromatography on hydroxyapatite.     

JC human papovavirus replication in human amnion cells.

JC human papovavirus was found to replicate in primary human amnion cells. The virus has undergone eight passages in amnion cells and was identified by serological methods as JC virus. By restriction endonuclease analysis of the viral DNA, the fragments observed were identical to those previously reported for the prototype strain.     

BK virus DNA sequence coding for the t and T antigens and evaluation of methods for determining sequence homology.

The DNA sequence of the early region of the human papovavirus BK (MM strain) was determined. A potential initiation signal for translation is located at nucleotides 3,047 to 3,045 or map position 0.614. Extending counterclockwise from this AUG signal there is only one open reading frame, which can code for a putative t antigen of 100 amino acids in length. If the early mRNA of BKV is spliced, then the regions between nucleotides 3,047 to 2,808 and 2,725 to 884 can code for a T antigen 694 amino acids in length. The sequences of the deduced T antigens in BK virus share 71% amino acid homology with those in simian virus 40, whereas the coding sequences of the two viruses share 70% DNA homology. Comparison of DNA sequences and evaluation of homology measurements between these two viruses are discussed.     

Lymphotropic papovavirus transformation of hamster embryo cells

Hamster embryo cells were transformed by African green monkey lymphotropic papovavirus (LPV). The transformed cells contained intranuclear T-antigens demonstrable by fluorescent antibody staining with hamster anti-LPV serum. Analysis of uncloned and cloned lines of transformed cells for LPV sequences revealed that the viral DNA was present as free nonintegrated and integrated genomes; there were approximately 10 copies of free DNA and about one to two copies of integrated genomes per cell. The cells were highly tumorigenic when inoculated into hamsters and produced progressively growing tumors in 100% of newborn or 10-day-old hamsters that were inoculated with LPV-transformed cells. The serum from tumor-bearing hamsters reacted with LPV-transformed cells and also showed a weak reaction with simian virus 40-, BK virus-, and JC virus-transformed cells, thereby showing an antigenic relationship with the T-antigens of other primate polyomaviruses. The large T-antigen of LPV was found to be an 84,000-molecular-weight protein which was immunoprecipitated by hamster anti-LPV (antiviral) as well as by tumor serum.