Complete Genome Sequence of a Novel Umbra-like Mycovirus From the Plant Pathogenic Fungus Phoma Matteucciicola

Here, a novel umbra-like mycovirus, ‘Phoma matteucciicola RNA virus 2’ (PmRV2), isolated from Phoma matteucciicola strain HNQH1 in Hainan province of China, was sequenced and analyzed. The complete genomic sequence of PmRV2 is 3,460 nucleotides (nts) with a GC content of 56.71%. Sequence analysis of PmRV2 indicated that the presence of two noncontiguous open reading frames (ORFs) encoding a hypothetical protein and a RNA-dependent RNA polymerase (RdRp), respectively. PmRV2 contains a metal-binding ‘GDN’ triad in Motif C of RdRp while most + ssRNA mycoviruses contained a ‘GDD’ motif in the same region. Additionally, a BLASTp search showed that the RdRp amino acid sequence of PmRV2 was most closely related to the RdRp of Macrophomina phaseolina umbra-like virus 1 (50.72% identity) and Erysiphe necator umbra-like virus 2 (44.84% identity). Phylogenetic analysis indicated that PmRV2 grouped together with Erysiphe necator umbra-like virus 2 (EnUlV2) within the recently proposed family of ‘Mycotombusviridae’.

Four Novel Botourmiaviruses Co-Infecting an Isolate of the Rice Blast Fungus Magnaporthe oryzae

Via virome sequencing, six viruses were detected from Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae, one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae. Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3′ untranslated region (3′-UTR) and 5′-UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae. Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 (MOBV5, MOBV6, and MOBV7), respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae, and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae.

Classical swine fever virus NS5B protein suppresses the inhibitory effect of NS5A on viral translation by binding to NS5A

In order to investigate molecular mechanisms of internal ribosome entry site (IRES)-mediated translation in classical swine fever virus (CSFV), an important pathogen of pigs, the expression level of NS3 was evaluated in the context of genomic RNAs and reporter RNA fragments. All data showed that the NS5A protein has an inhibitory effect on IRES-mediated translation and that NS5B proteins suppress the inhibitory effect of NS5A on viral translation, but CSFV NS5B GDD mutants do not. Furthermore, glutathione S-transferase pull-down assay and immunoprecipitation analysis, associated with deletion and alanine-scanning mutations, were performed. Results showed that NS5B interacts with NS5A and that the region aa 390–414, located in the C-terminal half of NS5A, is important for binding of NS5B to NS5A. Furthermore, amino acids K399, T401, E406 and L413 in the region were found to be essential for NS5A–NS5B interaction, virus rescue and infection. The above-mentioned region and four amino acids were observed to overlap with the site responsible for inhibition of IRES-mediated translation by the NS5A protein. We also found that aa 63–72, aa 637–653 and the GDD motif of NS5B were necessary for the interaction between NS5A and NS5B. These findings suggest that the repression activity of the NS5B protein toward the role of NS5A in translation might be achieved by NS5A–NS5B interaction, for which aa 390–414 of NS5A and aa 63–72, aa 637–653 and the GDD motif of NS5B are indispensable. This is important for understanding the role of NS5A–NS5B interaction in the virus life cycle.

Molecular characterization of the plant virus genus Ourmiavirus and evidence of inter-kingdom reassortment of viral genome segments as its possible route of origin

Ourmia melon virus (OuMV), Epirus cherry virus (EpCV) and Cassava virus C (CsVC) are three species placed in the genus Ourmiavirus. We cloned and sequenced their RNA genomes. The sizes of the three genomic RNAs of OuMV, the type member of the genus, were 2814, 1064 and 974 nt and each had one open reading frame. RNA1 potentially encoded a 97.5 kDa protein carrying the GDD motif typical of RNA-dependent RNA polymerases (RdRps). The putative RdRps of ourmiaviruses are distantly related to known viral RdRps, with the closest similarity and phylogenetic affinity observed with fungal viruses of the genus Narnaviridae. RNA2 encoded a 31.6 kDa protein which, expressed in bacteria as a His-tag fusion protein and in plants through agroinfiltration, reacted specifically with antibodies made against tubular structures found in the cytoplasm. The ORF2 product is significantly similar to movement proteins of the genus Tombusviridae, and phylogenetic analysis supported this evolutionary relationship. The product of OuMV ORF3 is a 23.8 kDa protein. This protein was also expressed in bacteria and plants, and reacted specifically with antisera against the OuMV coat protein. The sequence of the ORF3 protein showed limited but significant similarity to capsid proteins of several plant and animal viruses, although phylogenetic analysis failed to reveal its most likely origin. Taken together, these results indicate that ourmiaviruses comprise a unique group of plant viruses that might have evolved by reassortment of genomic segments of RNA viruses infecting hosts belonging to different eukaryotic kingdoms, in particular, fungi and plants.

Identification of amino acid sequences determining interaction between the cucumber mosaic virus-encoded 2a polymerase and 3a movement proteins

The cucumber mosaic virus (CMV)-encoded 3a movement protein (MP) is indispensable for CMV movement in plants. We have previously shown that MP interacts directly with the CMV-encoded 2a polymerase protein in vitro. Here, we further dissected this interaction and determined the amino acid sequences that are responsible for the MP and 2a polymerase protein interaction. Both the N-terminal 21 amino acids and the central GDD motif of the 2a polymerase protein were important for interacting with the MP. Although each of the regions alone was sufficient for the interaction with MP, quantitative yeast two-hybrid analyses showed that they acted synergistically to enhance the binding affinity. The MP N-terminal 20 amino acids were sufficient for interacting with the 2a polymerase protein, and the serine residue at position 14 played a critical role in the interaction. Multiple sequence alignment showed that the 2a protein interacting regions and the serine at position 14 in the MP are highly conserved among subgroup I and II CMV isolates.

Coordinate Replication of Alfalfa Mosaic Virus RNAs 1 and 2 Involves cis- and trans-Acting Functions of the Encoded Helicase-Like and Polymerase-Like Domains

ABSTRACT RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and coat protein. Transient expression of wild-type (wt) and mutant viral RNAs and proteins by agroinfiltration of plant leaves was used to study cis- and trans-acting functions of the helicase-like domain in P1 and the polymerase-like domain in P2. Three mutations in conserved motifs of the helicase-like domain of P1 affected one or more steps leading to synthesis of minus-strand RNAs 1, 2, and 3. In leaves containing transiently expressed P1 and P2, replication of wt but not mutant RNA 1 was observed. Apparently, the transiently expressed P1 could not complement the defect in replication of the RNA 1 mutant. Moreover, the transiently expressed wt replicase supported replication of RNA 2, but this replication was blocked in trans by coexpression of mutant RNA 1. However, expression of mutant RNA 1 did not interfere with the replication of RNA 3 by the wt replicase. Similarly, a mutation in the GDD motif encoded by RNA 2 could not be complemented in trans and affected the replication of RNA 1 by a wt replicase, while replication of RNA 3 remained unaffected. In competition assays, the transient wt replicase preferentially replicated RNA 3 over RNAs 1 and 2. The results indicate that one or more functions of P1 and P2 act in cis and point to the existence of a mechanism that coordinates the replication of RNAs 1 and 2.

Delayed Resistance to Plum Pox Potyvirus Mediated by a Mutated RNA Replicase Gene: Involvement of a Gene-Silencing Mechanism

Nicotiana benthamiana plants were transformed with intact and mutated nuclear inclusion b (NIb) gene sequences of plum pox potyvirus, Rankovic isolate (PPV-R). The constructs included additional in-frame initiation and termination codons. All of the eight independently transformed lines showed some kind of protection against PPV; however, this protection was largely overcome at high inoculum doses. Interestingly, a phenotype of total recovery from the initial infection was observed in a high percentage of plants of two lines transformed with an NIb coding sequence that carried a Gly to Val mutation at the GDD motif typical of viral RNA replicases. Recovery frequently started with the emergence of dark green patches in the infected leaves that accumulated much less virus than the surrounding tissue. Newly developing leaves were symptomless and virus free, and showed highly effective, and very specific, resistance to virus reinoculation. Both in the dark green patches and in the symptomless leaves, virus decline was accompanied by a drastic reduction in the accumulation of transgene transcripts. Normal transgene mRNA levels and initial susceptibility to infection were recovered in the progeny plants coming from the autofecundation of a recovered plant, indicating that the recovery phenotype is not meiotically stable. The results are discussed in terms of a previously proposed model that links gene silencing and RNA-mediated virus resistance (J. J. English, E. Mueller, and D. C. Baulcombe, Plant Cell 8:179–188, 1996), adapted to explain the recovery phenomenon.

Complementation and disruption of viral processes in transgenic plants

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode the replicase genes P1 and P2, respectively, whereas RNA 3 encodes the movem ent protein and the viral coat protein (CP). To investigate the mechanism of cross-protection, tobacco plants were transformed with wild-type and mutant DNA copies of the AIMV CP gene and the two replicase genes P1 and P2. Expression of wild-type CP at relatively low levels resulted in a resistance against infection with AIMV virus particles whereas at higher expression levels CP protected against infection with either AIMV particles or RNAs. Plants transformed with a mutant AIMV CP gene were not resistant to the wild-type virus but were resistant to AIMV with the same mutation in the CP gene. Transformation of plants with the wild-type P1 gene (P1 plants), P2 gene (P2 plants) or both these genes (P12 plants) did not result in resistance to AIMV. Instead, these plants could be infected with an inoculum lacking the gene(s) that was (were) integrated in the plant genome. Infection of non-transgenic plants, P1 plants or P2 plants with a mixture of AIMV genomic RNAs requires the presence of CP in the inoculum but P12 plants could be infected with RNA3 without any requirement for CP in the inoculum. Infection conditions in which 355 promoter/AlMV cDNA fusions were present in the inoculum instead of in the plant genome were used to shed light on the early function of CP. Finally, plants were transformed with P2 genes with mutations in the GDD-motif. A number of these transgenic lines showed a high level of resistance to AIMV.