Robert F. Bozarth

Structure and heterologous expression of the Ustilago maydis viral toxin KP4

Killer toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species, in the best‐characterized cases, they function by creating new pores in the ceil membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double‐stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double‐stranded RNA but differs from other known killer toxins in several respects: it has no N‐linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and h Is not processed by any of the known secretory protelnases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.

Structure and function of a virally encoded fungal toxin from Ustilago maydis: a fungal and mammalian Ca2+ channel inhibitor

BACKGROUND The P4 strain of the corn smut fungus, Ustilago maydis, secretes a fungal toxin, KP4, encoded by a fungal virus (UMV4) that persistently infects its cells. UMV4, unlike most other (non-fungal) viruses, does not spread to uninfected cells by release into the extracellular milieu during its normal life cycle and is thus dependent upon host survival for replication. In symbiosis with the host fungus, UMV4 encodes KP4 to kill other competitive strains of U. maydis, thereby promoting both host and virus survival. KP4 belongs to a family of fungal toxins and determining its structure should lead to a better understanding of the function and evolutionary origins of these toxins. Elucidation of the mechanism of toxin action could lead to new anti-fungal agents against human pathogens. RESULTS We have determined the atomic structure of KP4 to 1.9 A resolution. KP4 belongs to the alpha/beta-sandwich family, and has a unique topology comprising a five-stranded antiparallel beta-sheet with two antiparallel alpha-helices lying at approximately 45 degrees to these strands. The structure has two left-handed beta alpha beta cross-overs and a basic protuberance extending from the beta-sheet. In vivo experiments demonstrated abrogation of toxin killing by Ca2+ and, to a lesser extent, Mg2+. These results led to experiments demonstrating that the toxin specifically inhibits voltage-gated Ca2+ channels in mammalian cells. CONCLUSIONS Similarities, although somewhat limited, between KP4 and scorpion toxins led us to investigate the possibility that the toxic effects of KP4 may be mediated by inhibition of cation channels. Our results suggest that certain properties of fungal Ca2+ channels are homologous to those in mammalian cells. KP4 may, therefore, be a new tool for studying mammalian Ca2+ channels and current mammalian Ca2+ channel inhibitors may be useful lead compounds for new anti-fungal agents.

The molecular weight and packaging of dsRNAs in the mycovirus from Ustilago maydis killer strains.

The mycoviruses of Ustilago maydis killer strains are isometric, 43 nm in diameter, and contain several dsRNA segments designated heavy (H), medium (M), and light (L) according to their relative size. To determine the number of dsRNA segments per virion, major sedimenting and density components were isolated, their molecular weights determined from hydrodynamic properties, and their dsRNA contents determined by electron microscopy and/or polyacrylamide gel electrophoresis. The H dsRNA segments of 2.9, 3.1, and 4.2 x 10(6) daltons are separately encapsidated in isometric capsids that band in CsCI at 1.383, 1.394, and 1.418 g/cm8, respectively. The P1 strain contains the 3.1 and 4.2 x 10(6)-dalton segments, and the 3103 strain contains the 2.9 and 4.2 x 106-dalton segments. The T-4 strain contains the 3.1 x 106-dalton H segment and two M segments of 0.67 and 0.60 x 10(6) daltons. The H segments are separately encapsidated in virions which banded at 1.394 g/cm8, whereas the M segments are encapsidated in sets of one, two, or three in virions which banded at 1.314, 1.341, and 1.370 g/cm8. Molecular weights of 9.8 and 13.0 x 106 daltons were calculated for empty capsids (pCsCl = 1.278 g/cm8) and capsids containing the 3.1 x 10(6)-dalton dsRNA segments (pCsCl = 1.394 g/cm8). Analysis of components that banded at other densities in CsCl were consistent with the hypothesis that the banding pattern is the result of the encapsidation of a finite number of dsRNA segments in a capsid of 9.8 x 106 daltons. Although one to three M dsRNA segments were encapsidated in a single virion, no particles were detected with more than one H dsRNA segment per virion.

Biophysical and biochemical characterization of virus-like particles containing a high molecular weight ds-RNA from Helminthosporium maydis

Abstract Spherical virus-like particles (VLPs_ isolated from Helminthosporium maydis ATCC 32450 are 48 in diameter and have three serologically identical components which have the following properties: s 20,w = 152, 212, and 283; ϱ (in CsCl) = 1.298, 1.378, and 1.438 g cm −3 ; percentage of RNA = 0, 17, and 32; and molecular weight = 14, 17, and 21 × 10 6 . The nucleic acid of the fastest sedimenting component is double-stranded and has the following properties: s 20,w = 20.69; T m = 79.6°; ϱ (in Cs 2 SO 4 ) = 1.6062 g cm −3. ; molecular weight = 6.3 ± 0.5 × 10 6 .

Purification and molecular properties of the toxin coded by Ustilagomaydis virus P4

The toxin from the P4 strain of Ustilago maydis was purified and characterized using a series of gel-filtration and ion-exchange columns. The apparent molecular weight of the purified toxin was estimated from gel electrophoresis to be 11.3 kd in the presence of 2-mercaptoethanol and 10.3 kd in the absence of 2-mercaptoethanol. Amino acid analysis indicated 12% basic amino acids, 14% acidic amino acids and 16% glycine. The toxin was also stable to filtration and repeated freezing at -20 degrees C and thawing.

In vitro translation of the major capsid polypeptide from Ustilago maydis virus strain P1

Double-stranded RNA (dsRNA) from Ustilago maydis virus strain P1 was translated in vitro using a nuclease-treated rabbit reticulocyte lysate system. Following heat denaturation of the H2 double-stranded RNA segment in 90% dimethyl sulfoxide and incubation in the cell free extract, a primary translation product was observed which showed the same molecular weight and co-migrated with viral coat protein on 10% SDS-polyacrylamide gels. The in vitro product of the H2 dsRNA segment could also be immunoprecipitated with antibodies prepared against viral coat protein. Limited proteolysis of the in vitro product and authentic viral coat protein using Staphylococcus aureus V8 protease produced similar peptide patterns on SDS gels. In vitro translation products from other dsRNA segments that make up the P1 viral genome could not be precipitated by antibody to viral coat protein. These results complement the genetic data that indicated that information for coat formation and maintenance was contained within the H segments of dsRNA.

Ustilago maydis virus P4 killer toxin: Characterization, partial amino terminus sequence, and evidence for glycosylation

The toxin from Ustilago maydis virus P4 was purified to homogeneity and characterized. The native molecular mass, using size-exclusion HPLC was estimated to be 7.2 kDa. The purified toxin was composed of a single subunit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under reduced and nonreduced conditions resulted in estimated molecular masses of 8.4 and 7.4 kDa, respectively. The purified toxin was found to be glycosylated when tested for carbohydrates using the phenol-sulfuric acid method, Schiffs base reagent, and a Glycan detection kit and when probed against different biotinylated lectins. Partial amino acid sequence analysis of the purified toxin indicated a free N-terminus, 16% glycine, and 23% basic amino acid residues. No homology was found to either the alpha or the beta subunit of the toxin encoded by U. maydis infected with the P6 virus.

The characterization and crystallization of a virally encoded Ustilago maydis KP4 toxin.

KP4 is a virally encoded and highly specific toxin that kills fungi closely related to the fungus Ustilago maydis. The toxin was purified and crystals were formed using ammonium sulfate as precipitant. The crystals belong to the space group P6(1)(5)22 and diffracted to approximately 2.2 A resolution. Circular dicroism spectroscopy suggests that the protein is predominantly comprised of beta-strands.

Synthesis and processing of killer toxin from Ustilagomaydis virus P4

The synthesis of toxin protein from Ustilago maydis virus (UmV) strain P4 was studied in vitro and in vivo. The protein synthesized in vitro and in vivo has a molecular weight of approximately 30 kd whereas the native toxin has a molecular weight of about 12 kd. In the presence of protease inhibitors and glycosylation inhibitors, toxin protein synthesized in vivo showed higher molecular weight products that could be immunoprecipitated with toxin antibodies. These results suggest that the UmV P4 toxin protein is synthesized as a preprotein, which upon processing results in the 12 kd secreted form toxin.

Identification and Comparison of Viral Genes Coding for Capsid Proteins of Ustilago maydis Virus

Summary Direct evidence linking the capsid protein to specific dsRNA segments from the three killer strains of Ustilago maydis virus (P1, P4, P6) is presented. The capsid proteins of the three strains cross-react immunologically, have similar mol. wt. and similar peptide maps after limited proteolysis. The capsid proteins from P1 and P4 were translated from their respective H2 dsRNA segments, whereas the capsid protein for P6 was translated from H1 dsRNA. These in vitro translation products were each precipitated by the antiserum to capsid proteins of all three strains, had similar mol. wt. and similar peptide maps. All in vitro translation products competed effectively with native capsid proteins of all of the three strains in immunocompetition assays. These results suggest that the three strains code for a similar capsid protein, and that the information for capsid protein resides in the H2 segment of strain P1 and P4, and in the H1 segment of strain P6.