Efficient Transcription of a Compact Nucleoprotein Complex Isolated from Purified Simian Virus 40 Virions

Abstract

Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis- N-N′ -tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP 1 and VP 2 in addition to the proteins found associated with the viral minichromosome, i.e., VP 3 and histones H 2 A, H 2 B, H 3 , and H 4 . Our results suggest that the association of VP 1 , VP 2 , or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the “early” SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP 1 or VP 2 or both enhances the transcriptional activity and suggest that these “late” viral proteins may play a role in the regulation of expression of the SV40 genome.

Accurate transcription of simian virus 40 chromatin in a HeLa cell extract

“…We have recently reported that extracellular SV40 viral chromatin, which is similar to transcriptionally active eucaryotic genes, has a "relaxed" chromatin structure which makes the viral DNA highly accessible to enzymatic activities (4, 5). The conversion of SV40 chromatin to the relaxed conformation occurs as intracellular 75S chromatin sequentially matures into a 200S previrion, a 240S nuclear virion, and finally an extracellular virion (4,5,11,23). These studies demonstrate that the maturation process is accompanied by significant changes in chromatin composition, including increased acetylation of histones H3 and H4, the loss of histone H1, and the specific association of nonhistone viral protein with the chromatin complex.…”

“…The lengths of all RNA transcril corrected by 5.6% to compensate for migration rates of RNA and DNA molecu gels. 5). Similar of 3,450, 1,600, and 1,460 nucleotides.…”

“…After 2 h of adsorption at 37°C, Eagle minimal essential medium supplemented with 2% fetal calf serum was added. When cell lysis was complete (approximately 9 days), the infected cells and supernatant were collected, and the virus was purified as previously described (5).…”

“…Preparation of chromatin complexes and conditions for in vitro RNA synthesis. Preparation of SV40 chromatin complexes was carried out by ethylene glycolbis(O-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-dithiothreitol dissociation of SV40 virions as described previously (4,5), except that the NaCl concentration was decreased to 100 mM. HeLa cell extracts were prepared by the method of Manley et al (26), using cells that were harvested at a cell concentration of 5 x 105 to 6 x 105 cells per ml instead of 8 x 105 cells per ml as originally described.…”

Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species

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