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

Abstract

Host range mutations that permit SV-40 to grow with increased efficiency on SV-40-resistant [African green] monkey [kidney CV-1, CV-1f and MA134] cells were positioned within the viral B/C gene by a mapping method that relies on the coupling of specific DNA fragments. Pairs of [EcoRI, BamHI and HpaII] restriction endonucleases that each cleave SV-40 DNA at only 1 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 host range and temperature-sensitive A mutations. The mutations in 3 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. Monkey cells can efficiently join appropriate pairs of restriction endonuclease fragments intracellularly to produce infectious genomes, and the reaction was partially characterized. The general utility of fragment coupling (in vitro and in vivo) and heteroduplex repair for constructing and analyzing multiple mutants of SV-40 is discussed.