Virus‐like particles in yeast: Isolation and infectivity

Abstract

Virus-like particles containing electron dense cores are seen in thin sections of intact and degenerated cells of a therniosensitive ( t s ) strain of Candida tropicalis. A particulate fraction not present in wild-type cells has been isolated from the ts cells disrupted by pressure. The particles are 80-120 nm in diameter. Empty particles with a central cavity are observed. The method of infecting mating pairs of Saccharomyces cerevisiae by partially purified particles is described.In the past few years viruses and virus-like particles (VLP) have been discovered in several genera of fungi and moulds (BANKS et al. Materials and methodsWe used Candida tropicalis N-30 from the collection of the Allunion Institute of Protein Synthesis and two strains of the PETERROFF Genetic Stocks of Saccharomyces cereviaiae: 3-32B-P735 adel-,, hisx lys, pho l-,,S and 7A-Pl92 a ade ,-,metA, hisx.The following liquid and solid media were employed: Complete YADP, minimal MM (INGE-VECIITOMOV 1971), MMV (MM supplemented with 2 mg biotine, 200 mg thiamine HCI, and 500 mg L-alanine per litre), and MMVB (MM with 0.03% methylene blue). Standard cultivation was carried out at 30 "C.15-17 g of wet yeast cells were disrupted in a French press. The mixture was neutralized t o p H 7.0-7.2 with dry Tris and then incubated overnight with 500 pg/ml DNAse (REANAL) and and 500 pg/ml RNAse (Leningrad Meat Centre). After incubation, the mixture was centrifuged for 10 min a t 4,000 g, the supernatant was then centrifuged in a SPINCO type rotor for 20 min at 12000 g. After discarding the pellet, the supernatant was centrifuged for 90 min at 105,000 g. The pellet was suspended in 0.005 M Tris-phosphate buffer pH 7.0 and subjected to an additional cycle of low and high speed centrifugation in the same rotor. Another method of VLP isolation consisted in saturating the cultural fluid with (NH,),SO,, with subsequent holding overnight at 8 "C, and centrifugation a t 10,000 g in a SHARPLES type rotor. The pellet was dialyzed against Tris-phosphate buffer (0.005 M, p H 7.0) and subjected to differential centrifugation according to the first method.The cells werc fixed in 2.5% glutaraldehyde buffered.with s-collidine or in 1.5% KMnO,, post-fixed in 1.0% osmium tetroxide in the same buffer, and gradually embedded in Epon 812 under vacuum after careful dehydration through a graded series of ethanol solutions. Ultrathin sections were cut using an LKB Ultratome, stained with 2.0% aqueous acetate and lead citrate solution, and examined under YEMB-LOOB electron microscope. Samples of the cell extract of VLP were dropped on formvar-coated grids with a carbon backing, drained, stained with 2% phosphotungstate a t pH 7.0, and examined under an JEM-7 electron microscope.