A Cellular Protein with an RNA-Binding Activity Co-purifies with Viral dsRNA from Mycovirus-Infected Helminthosporium victoriae

Soldevila, Ana I.; Havens, Wendy M.; Ghabrial, Said A.

doi:10.1006/viro.2000.0349

Cited by 27 publications

(26 citation statements)

References 22 publications

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“…strain Wis. Q176 (ATCC 10002) and a number of strains derived from strain NRRL 1951 (ATCC 9480)] has remained unchanged throughout the years since PcV was first isolated (Banks et al, 1969;Buck et al, 1971;Border et al, 1972;Wood & Bozarth, 1972;Nash et al, 1973;Buck & Girvan, 1977;Edmondson et al, 1984;this study). This is also true for other chrysoviruses isolated from different Penicillium species (Buck & Girvan, 1977;Wood et al, 1971) and from various strains of Helminthosporium victoriae (Sanderlin & Ghabrial, 1978;Soldevila et al, 2000;Ghabrial et al, 2002). The co-presence of all four segments in different fungal species and strains harbouring chrysoviruses and the stability of the dsRNA patterns support the contention that all four segments are essential for infection and that none is defective or satellite in nature (see below for additional discussion).…”

Section: Discussionmentioning

confidence: 56%

Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus

Jiāng

Ghabrial

2004

Journal of General Virology

112

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Molecular cloning and complete nucleotide sequencing of Penicillium chrysogenum virus (PcV) dsRNAs indicated that PcV virions contained four dsRNA segments with sizes of 3562, 3200, 2976 and 2902 bp. Each dsRNA segment had unique sequences and contained a single large open reading frame (ORF). In vitro translation of transcripts derived from full-length cDNA clones of PcV dsRNAs yielded single products of sizes similar to those predicted from the deduced amino acid sequences of the individual ORFs. Sequence similarity searches revealed that dsRNA1 encodes a putative RNA-dependent RNA polymerase. In this study, it was determined that dsRNA2 encodes the major capsid protein and that p4, encoded by dsRNA4, is virion-associated as a minor component. All four dsRNAs of PcV, like the genomic segments of viruses with multipartite genomes, were found to have extended regions of highly conserved terminal sequences at both ends. In addition to the strictly conserved 5′-terminal 10 nt, a second region consisting of reiteration of the sequence CAA was found immediately upstream of the AUG initiator codon. These (CAA) n repeats are reminiscent of the translational enhancer elements of tobamoviruses. The 3′-terminal 14 nt were also strictly conserved. As PcV and related viruses with four dsRNA segments (genus Chrysovirus) have not been previously characterized at the molecular level, they were provisionally classified in the family Partitiviridae, comprising viruses with bipartite genomes. This study represents the first report on molecular characterization of a chrysovirus and the results suggest the creation of a new family of mycoviruses with multipartite dsRNA genomes to accommodate PcV and related viruses.

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Section: Discussionmentioning

confidence: 56%

Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus

Jiāng

Ghabrial

2004

Journal of General Virology

112

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“…Such a processing event has been found in the capsid protein of Helminthosporium victoriae virus 190S (Hv190SV), which belongs to the family Totiviridae (Soldevila et al, 2000). The apparent molecular mass of p58 is notably smaller than those of the PcV (109 kDa) and Hv145SV (100 kDa) coat proteins.…”

Section: Discussionmentioning

confidence: 99%

Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae

Urayama

Kato

Suzuki

et al. 2010

Journal of General Virology

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Mycoviruses causing impaired growth and abnormal pigmentation of the host were found in the rice blast fungus, Magnaporthe oryzae. Four dsRNAs, dsRNA 1 (3554 bp), dsRNA 2 (3250 bp), dsRNA 3 (3074 bp) and dsRNA 4 (3043 bp), were detected in isolate S-0412-II 1a of M. oryzae. By picking up single conidia of S-0412-II 1a, cured strains of the fungus were isolated that had completely lost the mycovirus. The cured strains had normal mycelial growth and pigmentation, suggesting that this mycovirus modulates host traits. The buoyant densities of isometric virus particles (~35 nm diameter) containing these dsRNAs in CsCl ranged from 1.37 to 1.40 g cm "3 . The single ORF (3384 nt) of dsRNA 1 encoded a gene product highly homologous to the viral RNA-dependent RNA polymerase of members of the family Chrysoviridae. It is noteworthy that mycovirus S-0412-II 1a was detected not only in host cells but also in culture supernatant. Furthermore, abnormal aggregation of mycelia was observed after adding the mycoviruscontaining culture supernatant to an uninfected strain of M. oryzae and mycoviral dsRNAs were detectable from the aggregated mycelia. This novel dsRNA mycovirus was named Magnaporthe oryzae chrysovirus 1.

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“…Note that the faint band located between the fully shifted (top) and free (bottom) RNA bands (marked with *) in panel F was not consistently detectable when we repeated these experiments. difluoride (PVDF) membranes (47). The membranes were renatured at room temperature in a renaturation buffer (10 mM Tris-Hcl [pH 7.5], 1 mM EDTA, 50 mM NaCl, 0.1% Triton X-100, and 1ϫ Denhardt's reagent [44]) with three changes of buffer for 20 min each.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus

Rajendran

Nagy

2003

J Virol

114

142

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Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine-and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.Viral-coded replicase proteins are essential for replication of tombusviruses, similar to other positive-strand RNA viruses (20,32,45). Tomato bushy stunt virus (TBSV), the prototypical tombusvirus, codes for p33 and p92 replicase proteins (reviewed in reference 43), which are essential for TBSV replication (32, 45). Based on the presence of the signature motifs for RNA-dependent RNA polymerase (RdRp) within the Cterminal portion of p92, the predicted function of the p92 is to synthesize viral RNA progenies (29,45). The function(s) of p33 in TBSV replication is currently unknown.An interesting feature of tombusviruses is that the larger replicase protein is expressed from the genomic RNA via a ribosomal readthrough mechanism of the p33 termination codon (43,45). Due to the expression strategy, the N-terminal portion of p92 overlaps with p33. Both p33 and p92 have been proposed to be part of the TBSV replication complexes (45). Accordingly, Western blot analysis of the partially purified TBSV RdRp preparation revealed the presence of both p33 and p92 in the transcriptionally active RdRp fractions (J. Pogany and P. D. Nagy, unpublished data).Both replicase proteins are localized to membranous structures in infected cells-the putative sites of tombusvirus replication (8, 45). The membrane localization domains in the replicase proteins are present within the N-t...

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A Cellular Protein with an RNA-Binding Activity Co-purifies with Viral dsRNA from Mycovirus-Infected Helminthosporium victoriae

Cited by 27 publications

References 22 publications

Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus

Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus

Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae

Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus

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