Viruses in fungi: infection of yeast with the K1 and K2 killer viruses.

Abstract

We demonstrate here that yeast killer viruses, previously thought to be transmitted only by cytoplasmic mixing during division, mating, or other induced forms of cell fusion, are capable of extracellular transmission. Viral particles from standard K1 and K2 killer strains were used to inoculate sensitive cells of Saccharomyces cerevisiae, rendered competent by spheroplasting, lithium acetate treatment, or by natural mating. Viruses with double-stranded RNA (dsRNA) genomes are widely distributed in nature, found in a wide variety of hosts-namely, animals (1), plants (2), insects (3), fungi (4), and bacteria (5). They are associated with the production of toxins in fungi, with hypovirulence of plant pathogens, including the chestnut blight fungus, which has virtually eliminated the American chestnut, and with rotaviral infections, the major cause of childhood diarrhea. The killer phenomenon in Saccharomyces cerevisiae is unique among eukaryotic dsRNA viral systems in the detail with which interactions between viral and host components have been explored and in the actual number ofhost components known to be involved (6).Type MATERIALS AND METHODS Yeast Strains and Media. Strain GG100-14D (a his3 ura3-50 trpl phoS pho3) contains L1A and L1Bc and was derived from the mating of two nonkiller strains, DB4 (a ura3-50 pho3 phoS) and DB13-1A (a his3-532 trpl gal2 suc2). The tester strains K7.S1 (a arg9) and S6 (a/a) lack M1 dsRNA and are sensitive to yeast killer toxin. The virus donor strains K7 (a arg9)s and Y110 (a/a) contain L1A M1 and L2A L2BC M2, respectively, and are standard K1 and K2 killers. Strain K23.A (a ade2) is a killer strain containing L1A L1BC and M1, used here and in previous work as a control for dsRNA and virus protein determinations. YEPD, methylene blue, and minimal media are described elsewhere (9, 10). YEPD pH 6.5 medium is YEPD medium adjusted to a final pH of 6.5. Buffered YEPD pH 6.5 medium is YEPD medium buffered with 0.2 M Na2HPO4/citrate to pH 6.5.Preparation of Viral Particles. Virus preparations were made from cells of strains K7 and Y110 grown in YEPD medium by Zymolyase/glass bead cell disruption, clarification by centrifugation, and purification of virus on 10-14% (wt/vol) sucrose gradients as described in detail elsewhere (7) with the following exceptions. Cells were not treated with 2-mercaptoethanol but rather were harvested, washed in SEKS buffer (1 M sorbitol/0.1 M EDTA/0.1 M Na2SO3/0.8 M KCl), resuspended in the same buffer, and treated with Zymolyase-20T (Seikagaku Kogyo) at 0.25 mg/g of wet cells. Virus used for inoculating spheroplasts and LiOAc-treated cells was suspended in STC buffer (10 mM Tris-HCl, pH 7.5/1.2 M sorbitol/10 mM CaCl2). Virus preparations used for inoculating mating cells were suspended in PKE buffer (30 mM NaPO4, pH 7.6/150 mM KCI/10 mM EDTA). In both cases virus preparations were used within 1 day and were kept at 4°C. Virus preparations stored refrigerated for 3 weeks became noninfectious.Abbreviations: dsRNA, double-stranded RNA; ScV, Saccharomyc...

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

“…A complete list of mycoviruses of plant pathogenic fungi can be found in a recent review by Pearson et al [4]. Despite the limited data on mycoviruses of human pathogenic fungi, evidence is provided that mycoviruses do exist in various fungal genera that are pathogenic to humans [6][7][8][9][10][11][12][13][14][15][16][17][18]. The mycoviruses that are associated with fungal genera that can cause human fungal disease and are also formally known by the ICTV are listed in Table 1.…”

“…Infection by the extracellular route could thus far only be achieved in special experimental settings using protoplasts. Applying this method, purified viral particles belonging to the dsRNA families of Partitiviridae, Totiviridae, and Reoviridae successfully infected fungal protoplasts [10,28,29].…”

“…These isolated Reoviridae were quite stable and retained infectivity upon prolonged storage [29]. In contrast, Saccharomyces cerevisiae Totivirus preparations stored refrigerated for three weeks became non-infectious [10]. Thus, isolation, production, purity and stability of mycoviruses of interest remain to be assessed.…”

“…Although a filamentous basidiomycete fungus, Ustilago maydis, is known to secrete killer toxins encoded by cytoplasmic dsRNA mycoviruses that are lethal to mycovirus-free susceptible strains of the same fungus [37][38][39] [10,40]. In contrast to the S. cerevisiae toxins, killer toxins of nonSaccharomyces yeasts and especially those secreted by virus-infected killer strains of the yeasts H. uvarum and Z. bailii show a broad-spectrum antimycotic potential to human pathogenic strains, such as Candida albicans, Sporothrix schenkii, and Fusarium sp.…”

Yeast Positive-stranded Virus-like RNA Replicons

“…However, we believe that this controversy could be reconciled if we take their biological context into consideration. So far all known mycoviruses have no extracellular pathway of transmission, except for some opportunistic events (37). It means that these viruses do not need elaborated machineries for exit and re-entry into new host cells.…”