Regulation of Simian Virus 40 Transcription: Sensitive Analysis of the RNA Species Present Early in Infections by Virus or Viral DNA

Abstract

The discrete species of SV-40 RNA present very early in infection of [African green] monkey cells were examined with wild-type virus, with mutant tSA58 virus and with the corresponding DNA to distinguish between 2 classes of models for control of late transcription: positive control mediated by large-T [tumor] antigen and negative control mediated by a repressor protein associated with viral DNA in the virion. Total cytoplasmic or nuclear polyadenylated RNA from infected cells were denatured with glyoxal, separated by electrophoresis on agarose gels, and transferred to diazobenzyloxymethyl paper. The positions of specific early and late RNA species were determined with region-specific SV-40 DNA probes. The technique can detect indvidual RNA present at the level of less than 1 copy/cell. After 9.5 h at 37.degree. C, appreciable amounts of 2 early RNA (2.6 kilobases [kb] and 2.9 kb) were present in the cytoplasm of cells infected with wild-type virus or DNA, along with much smaller amounts of 2 late RNA, 1.6 kb (16S) and 2.5 kb (19S). The amounts of the late RNA were reduced, but they were still synthesized in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. In comparable infections with tSA58 virus or DNA at nonpermissive temperature (41.degree. C), substantial amounts of the 2 early RNA were again present, but the 2 late RNA could not be detected. Small amounts of the late RNA were found when infections with tSA58 virus or DNA were prolonged to 30 h at 41.degree. C. These results are not consistent with negative control of late transcription through the action of a repressor and, taken together with other data, suggest that T antigen has an active role in late RNA synthesis. Specific early and late viral RNA were also detected in the nuclear poly(A)+ fractions and were similar in size to the RNA species found in the cytoplasmic polyadenylated fractions. The late nuclear RNA (1.8 and 2.9 kb) were significantly larger than the late cytoplasmic species, possibly because they are precursors. The 2.6 and 2.9 kb early RNA found in the cytoplasm are probably the messengers for large-T and small-t antigens, respectively.