Structure and assembly of double-stranded RNA mycoviruses.

Mycoviruses are a diverse group that includes ssRNA, dsRNA, and ssDNA viruses, with or without a protein capsid, as well as with a complex envelope. Most mycoviruses are transmitted by cytoplasmic interchange and are thought to lack an extracellular phase in their infection cycle. Structural analysis has focused on dsRNA mycoviruses, which usually package their genome in a 120-subunit T=1 icosahedral capsid, with a capsid protein (CP) dimer as the asymmetric unit. The atomic structure is available for four dsRNA mycovirus from different families: Saccharomyces cerevisiae virus L-A (ScV-L-A), Penicillium chrysogenum virus (PcV), Penicillium stoloniferum virus F (PsV-F), and Rosellinia necatrix quadrivirus 1 (RnQV1). Their capsids show structural variations of the same framework, with asymmetric or symmetric CP dimers respectively for ScV-L-A and PsV-F, dimers of similar domains of a single CP for PcV, or of two different proteins for RnQV1. The CP dimer is the building block, and assembly proceeds through dimers of dimers or pentamers of dimers, in which the genome is packed as ssRNA by interaction with CP and/or viral polymerase. These capsids remain structurally undisturbed throughout the viral cycle. The T=1 capsid participates in RNA synthesis, organizing the viral polymerase (1-2 copies) and a single loosely packaged genome segment. It also acts as a molecular sieve, to allow the passage of viral transcripts and nucleotides, but to prevent triggering of host defense mechanisms. Due to the close mycovirus-host relationship, CP evolved to allocate peptide insertions with enzyme activity, as reflected in a rough outer capsid surface.

Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF028.

Molecular characterization of the most common dsRNA element from Trichoderma atroviride indicated that it comprised 8,566 bp and encoded two large open reading frames (ORF1 and 2). The two ORFs were found to overlap by 46 bp with a typical (-1) slippery heptanucelotide sequence. The deduced protein sequences of ORF1 and ORF2 showed significant similarities to those of known mycoviral structural proteins and RNA-dependent RNA polymerases, respectively. Phylogenetic analysis indicated that this dsRNA is a member of a distinct species related to a group of unclassified mycoviruses; therefore, it was named Trichoderma atroviride mycovirus 1 (TaMV1).

Molecular characterization of a novel ssRNA ourmia-like virus from the rice blast fungus Magnaporthe oryzae.

In this study we characterize a novel positive and single stranded RNA (ssRNA) mycovirus isolated from the rice field isolate of Magnaporthe oryzae Guy11. The ssRNA contains a single open reading frame (ORF) of 2,373 nucleotides in length and encodes an RNA-dependent RNA polymerase (RdRp) closely related to ourmiaviruses (plant viruses) and ourmia-like mycoviruses. Accordingly, we name this virus Magnaporthe oryzae ourmia-like virus 1 (MOLV1). Although phylogenetic analysis suggests that MOLV1 is closely related to ourmia and ourmia-like viruses, it has some features never reported before within the Ourmiavirus genus. 3' RLM-RACE (RNA ligase-mediated rapid amplification of cDNA ends) and extension poly(A) tests (ePAT) suggest that the MOLV1 genome contains a poly(A) tail whereas the three cytosine and the three guanine residues present in 5' and 3' untranslated regions (UTRs) of ourmia viruses are not observed in the MOLV1 sequence. The discovery of this novel viral genome supports the hypothesis that plant pathogenic fungi may have acquired this type of viruses from their host plants.

Characterization of Botrytis cinerea negative-stranded RNA virus 1, a new mycovirus related to plant viruses, and a reconstruction of host pattern evolution in negative-sense ssRNA viruses.

The molecular characterization of a novel negative single-stranded RNA virus infecting the plant pathogenic fungus Botrytis cinerea is reported here. Comparison of the sequence of Botrytis cinerea negative-stranded RNA virus 1 (BcNSRV-1) showed a strong identity with RNA dependent RNA polymerases (RdRps) of plant pathogenic emaraviruses and tospoviruses. We have also found all the molecular signatures present in the RdRp of the genus Emaravirus and in other genera of family Bunyaviridae: the conserved TPD triplet and RY dinucleotide, the three basic residues in premotif A and the conserved motifs A, B, C, D, and E. Our results showed that BcNSRV-1 is phylogenetically close to members of the genus Emaravirus and of the family Bunyaviridae, and an ancestral state reconstruction using the conserved RdRp motifs of type members of each family of (-)ssRNA viruses indicated that BcNSRV-1 could possibly derive from an invertebrate and vertebrate-infecting virus.

Molecular characterization of Botrytis ourmia-like virus, a mycovirus close to the plant pathogenic genus Ourmiavirus.

The molecular characterization of a novel single-stranded RNA virus, obtained by next generation sequencing using Illumina platform, in a field grapevine isolate of the plant pathogenic fungus Botrytis, is reported in this work. The sequence comparison of this virus against the NCBI database showed a strong identity with RNA dependent RNA polymerases (RdRps) of plant pathogenic viruses belonging to the genus Ourmiavirus, therefore, this novel virus was named Botrytis ourmia-like virus (BOLV). BOLV has one open reading frame of 2169 nucleotides, which encodes a protein of 722 amino acids showing conserved domains of plant RNA viruses RdRps such as the most conserved GDD active domain. Our analyses showed that BOLV is phylogenetically closer to the fungal Narnavirus and the plant Ourmiavirus than to Mitovirus of the family Narnaviridae. Hence, we proposed that BOLV might represent the link between fungal viruses of the family Narnaviridae and the plant ourmiaviruses.

Specific binding of Fusarium graminearum Hex1 protein to untranslated regions of the genomic RNA of Fusarium graminearum virus 1 correlates with increased accumulation of both strands of viral RNA.

The HEX1 gene of Fusarium graminearum was previously reported to be required for the efficient accumulation of Fusarium graminearum virus 1 (FgV1) RNA in its host. To investigate the molecular mechanism underlying the production of FgHEX1 and the replication of FgV1 viral RNA, we conducted electrophoretic mobility shift assays (EMSA) with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that FgHex1 and both the 5'- and 3'-untranslated regions of plus-strand FgV1 RNA formed complexes. To determine whether FgHex1 protein affects FgV1 replication, we quantified accumulation viral RNAs in protoplasts and showed that both (+)- and (-)-strands of FgV1 RNAs were increased in the over-expression mutant and decreased in the deletion mutant. These results indicate that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding to the FgV1 genomic RNA.