Sequence properties
The results showed that M. oryzae strain TM02 was infected by a novel dsRNA virus, MoPmV1. Four distinct dsRNA segments with estimating sizes of 1.3–2.4 kb were detected in strain TM02 (Fig. 1A). The complete nucleotide sequences of the four genomic dsRNAs of MoPmV1 were determined with the length of 2421, 2280, 2018, and 1153 bp, and have been deposited in GenBank under the accession numbers MH231406.1-MH231409.1. Sequence analysis showed that dsRNA1 to dsRNA3 contained a large putative ORF (ORF1-ORF3) on the positive strand, while dsRNA4 contained two ORFs (ORF4a and ORF4b) (Fig. 1b). The 5’ terminus of each dsRNA starts with the conserved sequence “CGAACTTAAGAGTTTTTCT(A/G/C)CAC”, and the 3’ terminus of each segment ends with “ATTTTTAA” (Fig. 1C)..
dsRNA1 of MoPmV1contains contains one large ORF (ORF1, positions 36-2334), ORF1 encodes a 765-amino-acid (aa) protein with a predicted molecular mass of 83.4 kDa. The deduced aa sequence of ORF1contained eight conserved motifs (I-VIII) found in the picorna-like RdRp family of positive-strand, RNA eukaryotic viruses (RdRp-1, pfam00680) (Fig. 2). Concerning motif VI, like other polymycovirus, the GDD motif and the catalytic site of the RdRp is replaced with a GDNQ motif, which is similar to the negative-strand ssRNA viruses of the order Mononegavirales. A sequence search with BLASTP suggested that this protein was most closely related to the RdRps of some viruses in the genus polymycovirus, such as Beauveria bassiana polymycovirus 2 (BbPmV-2, GenBank accession number CUS18599.1, with identity of 59.2%, coverage of 98%, and E-value of 0, best hit), and Phaeoacremonium minimum tetramycovirus 1 (PmTmV-1, GenBank accession number QDB74985.1, with identity of 52.5%, coverage of 95%, and E-value of 0).
The sequence of dsRNA2 contained one large ORF (ORF2, positions 72-2163), which encodes a 696-aa protein with a molecular mass of 74.2-kDa with an unknown function (Fig. 1B). A BLASTp search for this protein gives hits for proteins from the same viruses that were identified in the ORF1 search. The first two of these hits were again proteins from PmTmV-1 (GenBank accession number QDB74986.1, with identity of 42.3%, coverage of 99%, and E-value of 1e-165, best hit) and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1, GenBank accession number CDP74619.1, with identity of 39.5%, coverage of 100%, and E-value of 4e-156).
The sequence of dsRNA3 contained one large ORF (ORF3, positions 55-1891), which encodes a 611-aa protein with a molecular mass of 64.8-kDa that is related to methyl-transferases of polymycoviruses (Fig. 1B). The first two hits of BLASTp were proteins from AfTmV1( CDP74620.1, with identity of 40.6%, coverage of 98%, and E-value of 5e-120, best hit) and Aspergillus fumigatus polymycovirus 1 (AfuPmV-1, GenBank accession number YP_009551546.1, with the identity of 40.4%, coverage of 93%, and E-value of 1e-115). In addition, an S-adenosylmethionine-dependent methyltransferase domain (SAM or AdoMet-MTase, cd02440, E-value = 3.47e-04) was found in the ORF3a-encoded protein at residues 134–250. As suggested for other polymycoviruses, this protein is probably involved in the capping of the 5’ terminuses of the positive strand of viral dsRNAs, which was proven experimentally in Beauveria bassiana polymycovirus 1 (BbPmV-1) [22].
The sequence of dsRNA4 contained two ORFs (ORF4a with positions 159–948 and ORF4b with positions 1014–1324), ORF4a encodes a 262-aa protein with a molecular mass of 27.8-kDa (Fig. 1B). This protein is proline-alanine-serine-rich (PAS-rich protein, PASrp), where proline, alanine and serine represent 7.6%, 9.5% and 7.2% of the total aa sequence, respectively. The PASrp encoded by ORF4a of MoPmV1 shares low sequence similarity (30.7% identity, 80% coverage, E-value = 5e-19) with the capsid protein (CP) of Colletotrichum camelliae filamentous virus 1 (CcFV-1, GenBank accession number ASV63095.1), whose particles are filamentous [3]. Since repeated sucrose gradient ultra-centrifugation experiments defeated to obtain conventional virus-like particles from MoPmV1, we tend to consider that MoPmV1 do not form conventional viral particles and the PASrp encoded by MoPmV1 dsRNA4 might function as a tentative structural component to unconventionally coat MoPmV1 dsRNA genome, just like some other polymycoviruses. However, the PASrp of MoPmV1 shares higher sequence similarity with the PASrps of other polymycoviruses, such as Botryosphaeria dothidea virus 1 (BdRV1, YP_009342471.1, with identity of 41.7%, coverage of 88%, and E-value of 1e-44, best hit), AfuPmV-1 (GenBank accession number YP_009551544.1, 35.9% identity, 99% coverage, E-value = 5e-36), Penicillium digitatum polymycovirus 1 (PdPmV-1, GenBank accession number AYP71806.1, 34.1% identity, 64% coverage, E-value = 4e-21) and BbPmV-1 (GenBank accession number YP_009352878.1, 36.7% identity, 72% coverage, E-value = 3e-18), for which no true virions have been observed [4,6,18]. A BLASTp search for ORF4b gives no hit, which sugests that the coding product has no significant similarity to other proteins in the database.
To analyze the taxonomic position of MoPmV1, a phylogenetic tree was constructed using the using the aa sequences of RdRp regions of MoPmV1 and other 56 selected viruses related to the family Polymycoviridae, Caliciviridae, Astroviridae, Hadakavirudae and Partitiviridae. As shown in Fig. 3, the neighbor-joining tree strongly suggested that MoPmV1 is a new member of the genus Polymycovirus in the family Polymycoviridae (Fig. 3). It is worth noting that, the genomic dsRNA sequences of another polymycovirus from four M. oryzae also have been deposited in the GenBank databases with accession number MW752168.1 -MH231471.1.Since the genomic sequence was submitted later than MoPmV1, the virus was proposed as Magnaporthe oryzae polymycovirus 2 (MoPmV2). In addition, the four genomic dsRNA sequences of MoPmV2 were from four different M. oryzae strains and were obviously not complete, since the sequencing method was Illumina, and the terminal sequences may not be accurate yet.
In conclusion, this is the fist report of the complete genomic sequence of a novle mycovirus in the family Polymycoviridae virus infecting the rice blast fugus M. oryzae.