Structure of the components of the adenovirus-simian virus 40 hybrid population Ad2 ++HEY and reciprocal molecular interactions

Abstract

The heterogeneous adenovirus-simian virus 40 hybrid population Ad2 ++HEY consists of Ad2 † † Abbreviations used: Ad, adenovirus; SV40, simian virus 40; WT, wild-type. Hybrid particles in the Ad2 ++HEY population containing 0.45, 1.43 and 2.39 integrated SV40 genomes are referred to as HEY-0.4, HEY-1.4 and HEY-2.4, respectively. One unit of DNA length(Ad unit, SV40 unit, HEY unit) is equal to the complete length of the corresponding genome (WT Ad2, SV40, HEY-1.4). The nomenclature for restriction enzymes was proposed by Smith & Nathans (1973). p.f.u., plaque-forming units. helper virions, non-hybrid SV40 virions, and three defective hybrid virions (HEY-0.4, HEY-1.4 and HEY-2.4) whose genomes differ in SV40 DNA content (0.45, 1.43 and 2.39 SV40 genomes, respectively). These components were purified by centrifugation in a CsCl gradient. The gradient fractions were identified and the structure of the hybrid DNA molecules was examined by using electron microscope heteroduplex methods. Furthermore, structural information was obtained by analyzing the DNA fragments resulting from digestion of hybrid DNA with restriction endonucleases. In HEY-1.4 and HEY-2.4 hybrids, which contain more than one complete SV40 genome, the excess SV40 DNA is organized as a head-to-tail tandem repetition. It was found that purified HEY-1.4 and HEY-2.4 hybrids interconvert readily in African green monkey kidney cells in the presence of Ad2 helper. Thus, the original composition of the population is restored by recombinational events. Also, HEY-2.4 could be cloned with Ad5 as helper, but similar experiments with HEY-1.4 and Ad5 only gave progeny in which part of the Ad2 information in. the hybrid genome had been exchanged for Ad5 sequences. Apparently, encapsidation by proteins, which are largely Ad5-specific, requires adapted structural features of the hybrid genomes. Infectious, non-hybrid SV40 particles are efficiently generated by the Ad2 ++HEY population. It was shown that HEY-2.4 hybrids yielded infectious non-hybrid SV40 virions much more efficiently than did HEY-1.4 hybrids. Excision of non-hybrid SV40 DNA is a very accurate process resulting in a homogeneous population of DNA molecules. No evidence was found for the presence of Ad or cellular DNA sequences in the excised nonhybrid SV40 genomes. Although the genome of the non-hybrid SV40 progeny differed slightly from that of SV40 strain 776, there is no reason to believe that these changes are causally related to the integration and excision phenomena.