A comprehensive open reading frame phylogenetic analysis of isometric positive strand ssRNA plant viruses.

Rigorous large-scale whole genome comparisons are capable of providing more comprehensive and potentially more accurate descriptions of viral relationships, allowing for the effective validation and modification of current taxonomy. Using a set of 5 togaviruses as an outgroup, a comprehensive phylogeny for 115 isometric positive ssRNA plant viruses was generated based on the simultaneous comparison of over 480 ORFs found within completely sequenced genomes. With the exception of a diverse group of viruses representing the family Comoviridae, the single tree generated contained well supported branches corresponding to well established groups of viruses, including Bromoviridae, Umbravirus, Sobemovirus, and Tymoviridae. In addition, evidence for specific relationships between groups were also observed, specifically Tombusviridae + Umbravirus, and Luteoviridae + Sobemovirus. Various well established subgroups of viruses were also well resolved within the tree. In addition, some recent proposals involving the creation of new genera or the inclusion of newly described viruses into established genera were supported, while others were not. The evidence for frequent gene sharing and the potential consequences to viral taxonomy are discussed.

A whole genome perspective on the phylogeny of the plant virus family Tombusviridae.

Most current classifications of viruses are based on single gene analysis of capsid protein or polymerase. The comparison of entire genomes is a more balanced approach that should provide a more complete picture of relatedness. We have used a singular value decomposition (SVD)-based analysis to generate phylogenetic trees using whole genome protein sequences from a family of single-stranded RNA plant viruses. Our dataset includes the 26 species of the family Tombusviridae, 25 of which have complete genome sequences cataloged in GenBank. The resulting phylogenetic tree agrees well with current taxonomic classifications, but with significant exceptions. One previously unassigned virus within this family, Maize necrotic streak virus, is definitively placed within the genus Tombusvirus by this analysis. In addition, the analysis defines two distinct subsets within the genus Necrovirus. Future datasets will be expanded to include other icosahedral positive strand RNA plant viruses, and then perhaps all positive strand RNA plant viruses.

RT-PCR Method for Detecting Cowpea Mottle Carmovirus in Vigna Germ Plasm.

A highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method was developed to detect cowpea mottle carmovirus (CPMoV) in newly acquired germ plasm of Vigna spp. It detected virus in tissues diluted up to 10-9. The preferred primers were designed from the RNA replicase cDNA sequence of CPMoV. These primers were able to detect CPMoV in plants infected with 10 different isolates of the virus. There were no cross-reactions with either bean mild mosaic or melon necrotic spot carmoviruses or any of the common cowpea viral pathogens tested. The RT-PCR method was up to 105 times more sensitive than direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA) in detecting CPMoV. The RT-PCR method gave no false positive reaction as is sometimes seen with ELISA.

The nucleotide sequence of cowpea mottle virus and its assignment to the genus Carmovirus.

The genome of cowpea mottle virus (CPMoV) is a positive ssRNA of 4029 nucleotides with six major open reading frames (ORFs). A non-coding region of 34 nucleotides precedes the first AUG. ORF1 encodes a 25 kDa polypeptide of unknown function and ORF2 encodes a 56 kDa putative RNA replicase. Like other members of carmoviruses, suppression of the amber termination codon of ORF1 would produce a readthrough polypeptide of 83 kDa. ORF3 and ORF4 encode two small proteins of 7.8 and 9.8 kDa, respectively. ORF5 encodes the 40 kDa capsid protein. ORF6 is located within ORF5 but is in a different frame and has no postulated function. CPMoV RNA is blocked at the 5' end and is not polyadenylated at the 3' end. Comparison of the physicochemical properties, genomic arrangement, and predicted amino acid sequences with those of other viruses justify the assignment of CPMoV to the genus Carmovirus, family Tombusviridae.

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.

Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of asymmetric Gag dimers.

The primary functions of most virus capsids are to protect the viral genome in the extra-cellular milieu and deliver it to the host. In contrast, the capsids of fungal viruses, like the cores of all other known double stranded RNA viruses, are not involved in host recognition but do shield their genomes, and they also carry out transcription and replication. Nascent (+) strands are extruded from transcribing virions. The capsids of the yeast virus L-A are composed of Gag (capsid protein; 76 kDa), with a few molecules of Gag-Pol (170 kDa). Analysis of these 420 A diameter shells and those of the fungal P4 virus by cryo-electron microscopy and image reconstruction shows that they share the same novel icosahedral structure. Both capsids consist of 60 equivalent Gag dimers, whose two subunits occupy non-equivalent bonding environments. Stoichiometry data on other double-stranded RNA viruses indicate that the 120-subunit structure is widespread, implying that this molecular architecture has features that are particularly favorable to the design of a capsid that is also a biosynthetic compartment.

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.

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 cell 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 it is not processed by any of the known secretory proteinases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.

Mapping and sequence analysis of the capsid protein gene of cowpea mottle virus.

Twelve cDNA clones were generated, covering approximately 95% of the cowpea mottle virus (CMeV) genome from the 3' end to near the 5' end. The entire capsid protein sequence of 1,104 nucleotides was contained in two clones located near the 3' terminus. The codons represented 367 amino acids (M(r) 39,611). The postulated amino acid sequence of CMeV capsid protein had 36% homology to turnip crinkle virus and 26% homology to carnation mottle virus in the arm and S domains, but western blots showed no serological relationship to either. On the basis of the organization and expression of its genome and its physicochemical properties, CMeV is assigned to the carmovirus group. Like other carmoviruses, CMeV generates three dsRNAs which are co-terminal at the 3' end in infected tissues, but CMeV differs from other carmoviruses in the absence of encapsidated subgenomic RNAs.

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, Schiff's 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.

Purification and molecular properties of the toxin coded by Ustilago maydis 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.

Semiconservative strand-displacement transcription of the M2 dsRNA segment of Ustilago maydis virus.

The P1 strain of the Ustilago maydis virus (UmV) is a segmented dsRNA virus with segments designated H1, H2, M1, M2, and L. Incubation of purified virus with a mixture of nucleotides containing 32P-UTP resulted in labeled dsRNA which was retained in the capsid and labeled ssRNA which was released from the capsid. This in vitro transcription reaction was dependent on Mg2+ ion and the optimum concentration for maximum incorporation was 10 mM. The pH and temperature optima were 8.0 and 30 degrees C, respectively. The ssRNA transcripts were precipitated from the supernatant solution of the reaction mixture after ultracentrifugation to separate the virus. Transcription products from supernatant solution hybridized with all five virion dsRNAs. Further studies of the M2 segment indicated that it was labeled within 2 h and the label was completely chased out in 2 h. Analysis of the labeled M2 dsRNA segment by strand-separation gel showed that only one strand (slow moving) was labeled. When both strands were tested in an in vitro translation system, only the slow-moving strand was translated to produce a 24 kDa product. Thus the M2 dsRNA segment of UmV P1 transcribes by a semiconservative strand-displacement mechanism.

Synthesis and processing of killer toxin from Ustilago maydis 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.

In vitro translation of the major capsid polypeptide from Ustilago maydis virus stain 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.

Six groups of double-stranded RNA mycoviruses.

Six groups of double-stranded (ds) RNA mycoviruses have been proposed. The main characteristics which define a group are described, and the properties of members and probable members of each group are tabulated. Possibilities for organization of the groups into families, genera and species are discussed. The classification scheme could ultimately accommodate the majority of the well-characterized dsRNA mycoviruses.

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.

Complex of Virus-Like Particles Containing Double-Stranded RNA from Thielaviopsis basicola.

Six randomly selected isolates of Thielaviopsis basicola were found to contain spherical virus-like particles (VLPs) approximately 40 nm in diameter. One isolate, ATCC 34114, selected for further analysis contained a complex of VLPs that sedimented as eight or more bands in sucrose density gradients and contained five size classes of double-stranded RNA. Five discrete precipitation lines were obtained in immunoelectrophoresis, which indicated that this isolate of T. basicola contains five distinct species of VLPs.