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.     

Transformation of hamster kidney cells by BK papovavirus DNA.

Supercoiled BK papovavirus DNA was shown to transform hamster kidney cells using the calcium phosphate co-precipitation technique. The transformed cells contained intranuclear T-antigen(s) and rescuable virus and produced progressively growing tumors when inoculated into hamsters. A novel finding was the production in tumor-bearing animals of antinuclear antibody, which reacted against normal, untransformed cells; in addition, tumor serum contained antibody against virus-specific T-antigen(s).     

Selective fluorescent labeling of amino groups of bovine pancreatic trypsin inhibitor by reductive alkylation

Bovine pancreatic trypsin inhibitor (BPTI) was reductively alkylated with 2-methoxy-1-naphthyl aldehyde and sodium cyanoborohydride (NaCNBH₃). All five possible derivatives, each labeled at one of the primary amino groups of BPTI, were obtained. The distribution of yields of the various derivatives can be controlled by changing the reaction conditions. Products were identified by high-performance liquid chromatography (HPLC) tryptic peptide mapping. This procedure was used for the preparation of three pure 2-methoxy-1-naphthyl-methylenyl-BPTI (MNA-BPTI) derivatives. Purification was achieved by means of affinity chromatography and HPLC. The spectral characteristics of the probe, notably monoexponential decay with a lifetime of 6.8 ± 0.1 ns and moderate limiting fluorescence polarization, P = 0.3 ± 0.015, make it a very useful donor in energy-transfer measurements.     

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.     

Uniform representation of the human papovavirus BK genome in transformed hamster cells.

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.     

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.     

Radioiodination of proteins by reductive alkylation

The use of the aliphatic aldehyde, para-hydroxyphenylacetaldehyde as the reactive moiety in the radioiodination of proteins by reductive alkylation is described. The para-hydroxyphenyl group is radiolabeled with 125I, reacted through its aliphatic aldehyde group with primary amino groups on proteins to form a reversible Schiff base linkage which can then be stabilized with the mild reducing agent NaCNBH3. The introduction of the methylene group between the benzene ring and the aldehyde group increases its reactivity with protein amino groups permitting efficient labeling at low aldehyde concentrations. Using this method, radioiodinated proteins with high specific activity can be produced. The reductive alkylation procedure is advantageous in that the labeling conditions are mild, the reaction is specific for lysyl residues, and the modification of the epsilon-ammonium group of lysine results in ionizable secondary amino groups avoiding major changes in protein charge.     

T antigen of BK papovavirus in infected and transformed cells.

BK virus T antigen from BKV-transformed rat and hamster cells and from productively infected monkey cells has been examined by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Forms of the antigen that migrate as proteins of 86,000 and 92,000 daltons have been identified. Both forms can be labeled by 32P.     

Iodination of DNA. Studies of the reaction and iodination of papovavirus DNA.

Iodination of DNA by the reaction originally described by S. L. Commerford ((1971), Biochemistry 10, 1993) is extremely sensitive to the secondary structure of the DNA. Cytidines in denatured simian virus 40 (SV40) DNA react at a slightly slower rate than free cytidine monophosphate; hydrogen-bonded cytidines in SV40 form I DNA are iodinated considerably more slowly; elimination of the negative supercoils in form I DNA by conversion to form II or form III reduces reactivity even further. The residual reactivity of form II or form III duplex DNA is not due to preferential iodination of unpaired cytidines near phosphodiester bond breaks; rather iodination occurs throughout the molecule. Cytidine monophosphate has been used as a model for DNA, to enable spectral measurements of its reaction with iodine and T1C13. At temperatures above 42 degrees C and at pH 5.0, formation of 5-iodocytidine is limited by the rate of formation of an intermediate, probably 5-iodo-6-hydroxydihydrocytidine. At lower temperatures, the conversion of intermediate to product is rate limiting, but can be accelerated by lowering the pH. By appropriate adjustment of pH, or temperature, the formation of intermediate or its conversion to product can be accelerated. Iodination destabilizes the DNA duplex. Iodocytosines in SV40 DNA are preferentially removed by S1 nuclease. Heavily iodinated DNA does not reassociate normally, but DNA with only 5-10% of its cytosines iodinated appears to reassociate with normal kinetics, if duplex formation is measured by hydroxylapatite chromatography. Conditions are described to permit preparation of DNA, which reassociates normally, having a specific activity of 10(8) cpm/mug.     

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.     

Presence of viral DNA sequences in hamster tumors induced by BK virus, a human papovavirus

DNA prepared from cell lines and transplanted tumors originating from five representative types of BKV-induced hamster tumors was examined for the presence of the BKV genome by analyzing DNA/DNA reassociation kinetics. BKV DNA sequences were detected in all cases. There were only a few (1--4) copies of BKV DNA per cell in one osteosarcoma and two ventricular tumors (one choroid plexus papilloma and one ependymoma), but there were multiple (up to 150) copies in one osteosarcoma, one ventricular tumor (choroid plexus papilloma), two insulinomas, one pineocytoma, and one cerebral neuroblastoma. In some cases the number of copies of the viral DNA differed among sister cell clones derived from the same primary tumor. Apparently some tumors contained nonintegrated free viral DNA besides the integrated BKV genome.     

Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus.

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.     

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.     

Expression of viral early functions in rat 3Y1 cells infected with human papovavirus BK.

A plaque morphology mutant (pm-522) of BK virus (BKV) with a small deletion at map unit 0.72 can readily transform rat 3Y1 cells, but wild-type BKV (wt-501) cannot. We examined the expression of the viral early functions in BKV (wt-501 or pm-522)-infected 3Y1 cells within a 2-week period after infection, before foci of transformed cells became detectable, to know how the difference between the two BKVs occurs. After a high-multiplicity infection, comparable amounts of free viral DNA (forms I and II) were found by Southern blotting analyses to persist in the nuclei of the cells infected with wt and pm BKVs. Whereas the proportion of T antigen-positive cells, as revealed by the indirect immunofluorescence method with complement, remained at a level of 60% in pm BKV infection, the level of T antigen-positive cells in wt BKV infection decreased from the initial 45% to 1% on day 9. The results obtained by the immunoprecipitation analyses of radiolabeled proteins from the infected cells were consistent with the immunofluorescence data. Viral early mRNA was detectable on day 2 and increased on day 9 in pm BKV infection, but in wt BKV infection, the low level of early mRNA detected on day 2 disappeared on day 9. Cell DNA synthesis and cell growth were enhanced more in pm BKV infection than in wt BKV infection. The low level of viral DNA synthesis that occurred in the infected rat cells was more prominent in pm BKV infection than in wt BKV infection. These data indicate that the expression of viral early functions continued much longer in pm BKV-infected rat cells than in wt BKV-infected rat cells, where the expression was probably repressed soon after infection. Continued T antigen production directed by the unintegrated viral genomes appears to be required for efficient transformation of rat cells by BKV.     

Immobilization of proteins by reductive alkylation with hydrophobic aldehydes

A new method is described for the immobilization of enzymes and other proteins onto hydrophobic gels. Trypsin, for example, can be quantitatively immobilized by reaction with sodium cyanoborohydride and dodecyladehyde-coated Octyl-Sepharose. Noncovalent interactions between the hydrophobic gel and approximately 10 resulting dodecylamino groups in the modified enzyme bind it very strongly but do not affect its ability to hydrolyze benzolarginine ethyl ester. The same procedure can also be used to immobilize E. Coli asparaginase and yeast alcohol dehydrogenase in high yield.