A neo-virus lifestyle exhibited by a (+)ssRNA virus hosted in an unrelated dsRNA virus: Taxonomic and evolutionary considerations

Recent studies illustrate that fungi as virus hosts provides a unique platform for hunting viruses and exploring virus/virus and virus/host interactions. Such studies have revealed a number of as-yet-unreported viruses and virus/virus interactions. Among them is a unique intimate relationship between a (+)ssRNA virus, yado-kari virus (YkV1) and an unrelated dsRNA virus, yado-nushi virus (YnV1). YkV1 dsRNA, a replicated form of YkV1, and RNA-dependent RNA polymerase, are trans-encapsidated by the capsid protein of YnV1. While YnV1 can complete its replication cycle, YkV1 relies on YnV1 for its viability. We previously proposed a model in which YkV1 diverts YnV1 capsids as the replication sites. YkV1 is neither satellite virus nor satellite RNA, because YkV1 appears to encode functional RdRp and enhances YnV1 accumulation. This represents a unique mutualistic virus/virus interplay and similar relations in other virus/host fungus systems are detectable. We propose to establish the family Yadokariviridae that accommodates YkV1 and recently discovered viruses phylogenetically related to YkV1. This article overviews what is known and unknown about the YkV1/YnV1 interactions. Also discussed are the YnV1 Phytoreo_S7 and YkV1 2A-like domains that may have been captured via horizontal transfer during the course of evolution and are conserved across extant diverse RNA viruses. Lastly, evolutionary scenarios are envisioned for YkV1 and YnV1

Three-Layered Complex Interactions among Capsidless (+)ssRNA Yadokariviruses, dsRNA Viruses, and a Fungus

We have previously discovered a virus neo-lifestyle exhibited by a capsidless positive-sense (+), single-stranded (ss) RNA virus YkV1 (family Yadokariviridae) and an unrelated double-stranded (ds) RNA virus YnV1 (proposed family "Yadonushiviridae") in a phytopathogenic ascomycete, Rosellinia necatrix. YkV1 has been proposed to replicate in the capsid provided by YnV1 as if it were a dsRNA virus and enhance YnV1 replication in return. Recently, viruses related to YkV1 (yadokariviruses) have been isolated from diverse ascomycetous fungi. However, it remains obscure whether such viruses generally show the YkV1-like lifestyle. Here, we identified partner viruses for three distinct yadokariviruses, YkV3, YkV4a, and YkV4b, isolated from R. necatrix that were coinfected with multiple dsRNA viruses phylogenetically distantly related to YnV1. We first established transformants of R. necatrix carrying single yadokarivirus cDNAs and fused them with infectants by single partner candidate dsRNA viruses. Consequently, YkV3 and YkV4s replicated only in the presence of RnMBV3 (family Megabirnaviridae) and RnMTV1 (proposed family "Megatotiviridae"), respectively. The partners were mutually interchangeable between the two YkV4 strains and three RnMTV1 strains but not between other combinations involving YkV1 or YkV3. In contrast to YkV1 enhancing YnV1 accumulation, YkV4s reduced RnMTV1 accumulation to different degrees according to strains. Interestingly, YkV4 rescued the host R. necatrix from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3) or host fungus. Overall, we revealed that while yadokariviruses generally require partner dsRNA viruses for replication, each yadokarivirus partners with a different dsRNA virus species in the three diverse families and shows a distinct symbiotic relation in a fungus. IMPORTANCE A capsidless (+)ssRNA virus YkV1 (family Yadokariviridae) highjacks the capsid of an unrelated dsRNA virus YnV1 (proposed family "Yadonushiviridae") in a phytopathogenic ascomycete, while YkV1 trans-enhances YnV1 replication. Herein, we identified the dsRNA virus partners of three yadokariviruses (YkV3, YkV4a, and YkV4b) with genome organization different from YkV1 as being different from YnV1 at the suborder level. Their partners were mutually interchangeable between the two YkV4 strains and three strains of the partner virus RnMTV1 (proposed family "Megatotiviridae") but not between other combinations involving YkV1 or YkV3. Unlike YkV1, YkV4s reduced RnMTV1 accumulation and rescued the host fungus from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3, family Megabirnaviridae) or host fungus. These revealed that while each yadokarivirus has a species-specific partnership with a dsRNA virus, yadokariviruses collectively partner extremely diverse dsRNA viruses and show three-layered complex mutualistic/antagonistic interactions in a fungus

A capsidless ssRNA virus hosted by an unrelated dsRNA virus

Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication1. Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix2. YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses3, while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses4. Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus

Identification of an RNA Silencing Suppressor Encoded by a Symptomless Fungal Hypovirus, Cryphonectria Hypovirus 4

Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)—likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1

A transfectable Fusagravirus from a Japanese strain of Cryphonectria carpinicola with spherical particles

A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5′-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of ~40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection

Diverse Partitiviruses From the Phytopathogenic Fungus,Rosellinia necatrix

Partitiviruses (dsRNA viruses, familyPartitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus,Rosellinia necatrix, only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16R. necatrixstrains belonging to 15 new species, for which "Rosellinia necatrix partitivirus 11-Rosellinia necatrix partitivirus 25" were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera,Alphapartitivirus, andBetapartitivirus. Some partitiviruses were transfected into reference strains of the natural host,R. necatrix, and an experimental host,Cryphonectria parasitica, using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns ofR. necatrixandC. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed

Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi

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

Exploration of the yadokari/yadonushi nature of YkV3 and RnMBV3 in the original host and a model filamentous fungus

The yadokari/yadonushi nature is a recently discovered virus lifestyle; “yadokari” refers to the ability of capsidless positive-sense (+) RNA viruses (yadokariviruses) to utilize the capsids of phylogenetically distant double-stranded RNA (dsRNA) viruses possibly as the replication site, while “yadonushi” refers to the ability of dsRNA viruses to provide capsids to yadokariviruses. This virus–virus interaction, however, has been only studied with limited pathosystems. Here, we established a new study model with a capsidless (+)RNA yadokarivirus YkV3 (family Yadokariviridae) and its capsid donor RnMBV3 (family Megabirnaviridae) in the original host fungus Rosellinia necatrix and a model filamentous fungal host Cryphonectria parasitica. YkV3 has a simple genome structure with one open reading frame of 4305 nucleotides encoding a single polyprotein with an RNA-dependent RNA polymerase and a 2A-like self-cleavage peptide domain. Reverse genetics of YkV3 in R. necatrix showed that YkV3 tolerates a nucleotide substitution in the extreme 5′-terminus. The insertion of two termination codons immediately downstream of the 2A-like cleavage site abolished YkV3 viability, suggesting the importance of the C-terminal portion of the polyprotein of unknown function. Transfection of RnMBV3 and YkV3 into an RNA silencing-deficient mutant Δdcl2 of C. parasitica showed the replication competency of both viruses. Comparison between the wild-type and Δdcl2 strains of C. parasitica in virus accumulation suggested that RnMBV3 and YkV3 are susceptible to RNA silencing in C. parasitica. Taken together, we have established a platform to further explore the yadokari/yadonushi nature using genetically manipulable host fungal and virus strains

A New Double-Stranded RNA Mycovirus in Cryphonectria naterciae Is Able to Cross the Species Barrier and Is Deleterious to a New Host

Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field-collected strains of C. naterciae, 40% tested CnFGV1-positive. Cocultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae upon virus infection. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe

Proof of Concept of the Yadokari Nature: a Capsidless Replicase-Encoding but Replication-Dependent Positive-Sense Single-Stranded RNA Virus Hosted by an Unrelated Double-Stranded RNA Virus

Viruses typically encode their own capsids that encase their genomes. However, a capsidless positive-sense single stranded RNA [(+)ssRNA] virus, YkV1, depends on an unrelated double-stranded RNA (dsRNA) virus, YnV1, for encapsidation and replication