The structure and expression of two defective adenovirus 2/simian virus 40 hybrids

Abstract

Two new defective adenovirus 2/simian virus 40 hybrids (Ad2 + D1 and Ad2 + D2) have been isolated from the population known as Ad2 ++ HEY (Lewis & Rowe, 1970). The structure of their genomes has been determined by analysis with restriction enzymes, hybridization and electron microscopy. The DNA of Ad2 +D1 is almost equal in size to that of its helper, adenovirus 2. It contains an insertion of simian virus 40 (SV40) sequences 3.2 × 10 3 bases long in place of the 3.5 × 10 3 base segment of adenovirus 2 DNA which maps between 0.64 and 0.74 fractional genome lengths from the left end of the viral DNA. The insertion which encompasses the entire early region of the SV40 genome, comprises the segment of DNA lying between positions 71 and 10: it is oriented with the sequences coding for the carboxy terminus of the A gene protein to the left. The DNA of Ad2 +D2 is shorter than that of adenovirus 2 by approximately 6%. The segment mapping between 0.76 and 0.96 fractional genome lengths from the left end of adenovirus 2 DNA is absent: in its place is inserted a stretch of DNA, 5.0 × 10 3 bases in length which comprises all the sequences of the SV40 genome apart from those lying between map positions 54 and 63. By contrast to Ad2 +D1, the ‘early’ SV40 sequences in Ad2 +D2 are transcribed from left to right. RNA complementary to the ‘early’ sequences of the E strand of SV40 DNA is present in the cytoplasm at both early and late times after infection of monkey cells by Ad2 +D1. No transcripts of the viral L strand were detected. SV40-specific RNA was found in the cytoplasm of cells infected with Ad2 +D2, only at late times after infection. The most abundant species was complementary to the coding sequences of the E strand of SV40 DNA. Transcripts of the L strand were also present, but in significantly lesser quantity. Proteins related to SV40-specific T antigen were detected in infected cells by immunofluorescence and by immunoprecipitation with sera from hamsters bearing tumors induced by SV40. The major polypeptide precipitated from extracts of cells infected with Ad2 +D1 migrated through sodium dodecyl sulfate/polyacrylamide gels at the same rate as SV40 T antigen: its apparent molecular weight is 96,000. A significantly larger polypeptide ( M r = 115,000) is precipitated from extracts of cells infected by Ad2 +D2. Consideration of the structures of the genomes of Ad2 +D1 and Ad2 +D2 leads us to believe that Ad2 +D1 may have arisen by recombination between members of the Ad2 ++HEY hybrid pool and adenovirus 2, while Ad2 +D2 was most likely generated by recombination events between adenovirus 2 and free SV40.