Genetic analysis of host range mutant viruses suggests an uncoating defect in simian virus 40-resistant monkey cells

Host range mutations that permit simian virus 40 (SV40) to grow with increased efficiency on SV40-resistant monkey cells have been positioned within the viral B/C gene by a mapping method that relies on the coupling of specific DNA fragments. Pairs of restriction endonucleases that each cleave SV40 DNA at only one site were used to generate pairs of specific DNA fragments. Corresponding pairs of fragments were purified from host range mutant and wild-type DNA and joined in known combinations to determine the location of the host range mutations. The map position of the host range mutations was confirmed by using the same technique to generate and couple genetically marked viral DNA fragments to produce the predicted double mutants. Three different double mutants were constructed that carry both host range and temperature-sensitive A mutations. The mutations in three independently isolated host range mutant viruses are located at very close, perhaps identical, sites, because no wild type viruses were produced from the cell-mediated repair of pairwise heteroduplexes between them. The location of these host range mutations suggests that their phenotype results from mutational alteration of the major capsid protein, the product of the B/C gene. In addition it was demonstrated that monkey cells can efficiently join appropriate pairs of restriction endonuclease fragments intracellularly to produce infectious genomes. That reaction has been partially characterized. The general utility of fragment coupling (in vitro and in vivo) and heteroduplex repair for constructing and analyzing multiple mutants of SV40 is discussed.