The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to ϳ50% for ⌬p29, and 10 to 20% for ⌬p69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for ⌬p29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a ⌬p69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys 70 or Cys 72 , strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.Members of the virus family Hypoviridae (22) persistently attenuate virulence (hypovirulence) and stably alter complex biological processes upon infection of their fungal host, the chestnut blight fungus, Cryphonectria parasitica. Infection-related phenotypic changes can include suppressed pigment production and asexual sporulation, altered colony morphology, loss of female fertility and modified expression of specific host genes (1,18,28,31). Infectious hypovirus cDNA clones (8, 10) and a robust transformation protocol for C. parasitica (15) provide a versatile system for identifying virus-encoded symptom determinants and modulators of hypovirus replication.Choi and Nuss (13) showed that transformation of virus-free C. parasitica with the 5Ј-proximal coding domain, open reading frame (ORF) A, resulted in suppressed conidiation and pigmentation but no change in fungal virulence. ORF A encodes a papain-like protease, p29, and a highly basic protein, p40, derived, respectively, from the N terminus and C terminus of polyprotein, p69, by a p29-mediated autocatalytic cl...

Insects are the host and vector of diverse viruses including those that infect vertebrates, plants, and fungi. Recent wide-scale transcriptomic analyses have uncovered the existence of a number of novel insect viruses belonging to an alphavirus-like superfamily (virgavirus/negevirus-related lineage). In this study, through an in silico search using publicly available insect transcriptomic data, we found numerous virus-like sequences related to insect virga/nege-like viruses. Phylogenetic analysis showed that these novel viruses and related virus-like sequences fill the major phylogenetic gaps between insect and plant virga/negevirus lineages. Interestingly, one of the phylogenetic clades represents a unique insect-infecting virus group. Its members encode putative coat proteins which contained a conserved domain similar to that usually found in the coat protein of plant viruses in the family Virgaviridae. Furthermore, we discovered endogenous viral elements (EVEs) related to virga/nege-like viruses in the insect genomes, which enhances our understanding on their evolution. Database searches using the sequence of one member from this group revealed the presence of EVEs in a wide range of insect species, suggesting that there has been prevalent infection by this virus group since ancient times. Besides, we present detailed EVE integration profiles of this virus group in some species of the Bombus genus of bee families. A large variation in EVE patterns among Bombus species suggested that while some integration events occurred after the species divergence, others occurred before it. Our analyses support the view that insect and plant virga/nege-related viruses might share common virus origin(s).

Mycoreovirus 1 (MYRV-1) is the type species of the newly described genus Mycoreovirus of the large virus family Reoviridae. The virus was isolated from a hypovirulent strain (9B21) of the chestnut blight fungus, Cryphonectria parasitica. A previous study showed that double-shelled particles introduced to fungal spheroplasts resulted in stably infected colonies. Of the 11 double-stranded RNA genomic segments (S1-S11), the three largest (S1-S3) were sequenced previously and shown to have moderate levels of similarity to the homologous segments of mammal-pathogenic coltiviruses (Eyach virus and Colorado tick fever virus) and another fungus-infecting reovirus, Mycoreovirus 3 of Rosellinia necatrix strain W370 (MYRV-3/RnW370). The sequences of the remaining segments (S4-S11) are reported here. All of the segments have single ORFs on their positive strands and the terminal sequences 59-GAUCA----GCAGUCA-39 are conserved among currently and previously sequenced segments. Oligo-cap analysis showed that the positive strands of the genomic segments are capped, whereas the negative strands are not. Similarities among the four evolutionarily related viruses include low or moderate levels of amino acid sequence identity (14?7-34?2 %) and isoelectric points among equivalent polypeptides, e.g. proteins encoded by segments S4 and S5 of the four viruses. Phylogenetic analysis indicated that MYRV-1/Cp9B21 is related more closely to MYRV-3/RnW370 than to the coltiviruses. An interesting dissimilarity is found in codon-choice pattern among the four viruses, i.e. MYRV-1/Cp9B21 segments have a lower frequency of [XYG+XYC] than corresponding segments of the other viruses, suggesting a possible adjustment of virus codon usage to their host environments.

SUMMARYMembers of the plant Dicer-like (DCL) protein family are the critical components of the RNA-silencing pathway that mediates innate antiviral defence. The distinct antiviral role of each individual DCL protein has been established with mostly based on observations of aerial parts of plants. Thus, although the roots are closely associated with the life cycle of many plant viruses, little is known about the antiviral activities of DCL proteins in roots. We observed that antiviral silencing strongly inhibits potato virus X (PVX) replication in roots of some susceptible Solanaceae species. Silencing of the DCL4 homolog in Nicotiana benthamiana partially elevated PVX replication levels in roots. In Arabidopsis thaliana, which was originally considered a non-host plant of PVX, high levels of PVX accumulation in inoculated leaves were achieved by inactivation of DCL4, while in the upper leaves and roots, it required the additional inactivation of DCL2. In transgenic A. thaliana carrying the PVX amplicon with a green fluorescent protein (GFP) gene insertion in the chromosome (AMP243 line), absence of DCL4 enabled high levels of PVX-GFP accumulation in various aerial organs but not in the roots, suggesting that DCL4 is critical for intracellular antiviral silencing in shoots but not in roots, where it can be functionally compensated by other DCL proteins. Together, the high level of functional redundancies among DCL proteins may contribute to the potent antiviral activities against PVX replication in roots.

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a -1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.