Penicillium Stoloniferum Virus Research Articles

Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1–3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.

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.

Capsid Structure of dsRNA Fungal Viruses.

Most fungal, double-stranded (ds) RNA viruses lack an extracellular life cycle stage and are transmitted by cytoplasmic interchange. dsRNA mycovirus capsids are based on a 120-subunit T = 1 capsid, with a dimer as the asymmetric unit. These capsids, which remain structurally undisturbed throughout the viral cycle, nevertheless, are dynamic particles involved in the organization of the viral genome and the viral polymerase necessary for RNA synthesis. The atomic structure of the T = 1 capsids of four mycoviruses was resolved: the L-A virus of Saccharomyces cerevisiae (ScV-L-A), Penicillium chrysogenum virus (PcV), Penicillium stoloniferum virus F (PsV-F), and Rosellinia necatrix quadrivirus 1 (RnQV1). These capsids show structural variations of the same framework, with 60 asymmetric or symmetric homodimers for ScV-L-A and PsV-F, respectively, monomers with a duplicated similar domain for PcV, and heterodimers of two different proteins for RnQV1. Mycovirus capsid proteins (CP) share a conserved α-helical domain, although the latter may carry different peptides inserted at preferential hotspots. Insertions in the CP outer surface are likely associated with enzymatic activities. Within the capsid, fungal dsRNA viruses show a low degree of genome compaction compared to reoviruses, and contain one to two copies of the RNA-polymerase complex per virion.

Molecular characterization of a new gammapartitivirus isolated from the citrus-pathogenic fungus Penicillium digitatum

To date, partitiviruses, including gammapartitiviruses, have been extensively studied in various fungal hosts but have not been reported in Penicillium digitatum (also called green mold, the pathogenic fungus infecting citrus). In the present work, we isolated and molecularly characterized a double-stranded RNA (dsRNA) partitivirus from citrus green mold, which we have named "Penicillium digitatum gammapartitivirus 1" (PdGV1). The bisegmented genome of PdGV1 contains two dsRNA segments (dsRNA1 and dsRNA2) with a length of 1795 bp and 1622 bp, respectively. Each of the two genomic dsRNAs contains a single open reading frame encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. Phylogenetic analysis based on RdRp and CP sequences showed that PdGV1 clustered with mycoviruses belonging to the genus Gammapartitivirus, family Partitiviridae, e.g., Penicillium stoloniferum virus S. The 5'- and 3'-untranslated regions (UTRs) of the PdGV1 genomic dsRNAs both contained unique conserved RNA motifs that have never been found in any other partitivirus. This is the first report of a new gammapartitivirus that infects the citrus-pathogenic fungus P. digitatum.

Using a Novel Partitivirus in Pseudogymnoascus destructans to Understand the Epidemiology of White-Nose Syndrome.

White-nose syndrome is one of the most lethal wildlife diseases, killing over 5 million North American bats since it was first reported in 2006. The causal agent of the disease is a psychrophilic filamentous fungus, Pseudogymnoascus destructans. The fungus is widely distributed in North America and Europe and has recently been found in some parts of Asia, but interestingly, no mass mortality is observed in European or Asian bats. Here we report a novel double-stranded RNA virus found in North American isolates of the fungus and show that the virus can be used as a tool to study the epidemiology of White-nose syndrome. The virus, termed Pseudogymnoascus destructans partitivirus-pa, contains 2 genomic segments, dsRNA 1 and dsRNA 2 of 1.76 kbp and 1.59 kbp respectively, each possessing a single open reading frame, and forms isometric particles approximately 30 nm in diameter, characteristic of the genus Gammapartitivirus in the family Partitiviridae. Phylogenetic analysis revealed that the virus is closely related to Penicillium stoloniferum virus S. We were able to cure P. destructans of the virus by treating fungal cultures with polyethylene glycol. Examination of 62 isolates of P. destructans including 35 from United States, 10 from Canada and 17 from Europe showed virus infection only in North American isolates of the fungus. Bayesian phylogenetic analysis using nucleotide sequences of the viral coat protein geographically clustered North American isolates indicating fungal spread followed by local adaptation of P. destructans in different regions of the United States and Canada. This is the first demonstration that a mycovirus potentially can be used to study fungal disease epidemiology.

A novel partitivirus that confers hypovirulence on plant pathogenic fungi.

Members of the family Partitiviridae have bisegmented double-stranded RNA (dsRNA) genomes and are not generally known to cause obvious symptoms in their natural hosts. An unusual partitivirus, Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), conferred hypovirulence on its natural plant-pathogenic fungal host, Sclerotinia sclerotiorum strain WF-1. Cellular organelles, including mitochondria, were severely damaged. Hypovirulence and associated traits of strain WF-1 and SsPV1/WF-1 were readily cotransmitted horizontally via hyphal contact to different vegetative compatibility groups of S. sclerotiorum and interspecifically to Sclerotinia nivalis and Sclerotinia minor. S. sclerotiorum strain 1980 transfected with purified SsPV1/WF-1 virions also exhibited hypovirulence and associated traits similar to those of strain WF-1. Moreover, introduction of purified SsPV1/WF-1 virions into strain KY-1 of Botrytis cinerea also resulted in reductions in virulence and mycelial growth and, unexpectedly, enhanced conidial production. However, virus infection suppressed hyphal growth of most germinating conidia of B. cinerea and was eventually lethal to infected hyphae, since very few new colonies could develop following germ tube formation. Taken together, our results support the conclusion that SsPV1/WF-1 causes hypovirulence in Sclerotinia spp. and B. cinerea. Cryo-EM (cryo-electron microscopy) reconstruction of the SsPV1 particle shows that it has a distinct structure with similarity to the closely related partitiviruses Fusarium poae virus 1 and Penicillium stoloniferum virus F. These findings provide new insights into partitivirus biological activities and clues about molecular interactions between partitiviruses and their hosts. Members of the Partitiviridae are believed to occur commonly in their phytopathogenic fungal and plant hosts. However, most partitiviruses examined so far appear to be associated with latent infections. Here we report a partitivirus, SsPV1/WF-1, that was isolated from a hypovirulent strain of Sclerotinia sclerotiorum and describe its biological and molecular features. We have demonstrated that SsPV1 confers hypovirulence. Furthermore, SsPV1 can infect and cause hypovirulence in Botrytis cinerea. Our study also suggests that SsPV1 has a vigorous ability to proliferate and spread via hyphal contact. SsPV1 can overcome vegetative incompatibility barriers and can be transmitted horizontally among different vegetative compatibility groups of S. sclerotiorum, even interspecifically. Cryo-EM reconstruction of SsPV1 shows that it has a distinct structure with similarity to closely related partitiviruses. Our studies exploit a novel system, SsPV1 and its hosts, which can provide the means to explore the mechanisms by which partitiviruses interact with their hosts.

Atomic structure reveals the unique capsid organization of a dsRNA virus

For most dsRNA viruses, the genome-enclosing capsid comprises 120 copies of a single capsid protein (CP) organized into 60 icosahedrally equivalent dimers, generally identified as 2 nonsymmetricallyinteracting CP molecules with extensive lateral contacts. The crystal structure of a partitivirus, Penicillium stoloniferum virus F (PsV-F), reveals a different organization, in which the CP dimer is related by almost-perfect local 2-fold symmetry, forms prominent surface arches, and includes extensive structure swapping between the 2 subunits. An electron cryomicroscopy map of PsV-F shows that the disordered N terminus of each CP molecule interacts with the dsRNA genome and probably participates in its packaging or transcription. Intact PsV-F particles mediate semiconservative transcription, and transcripts are likely to exit through negatively charged channels at the icosahedral 5-fold axes. Other findings suggest that the PsV-F capsid is assembled from dimers of CP dimers, with an arrangement similar to flavivirus E glycoproteins.

Genomic characterization of a novel partitivirus infecting Aspergillus ochraceus

Three double-stranded RNA (dsRNA) segments from Aspergillus ochraceus isolate FA 0611, designated as AoR1, AoR2, and AoR3, were cloned and sequenced. AoR1 was identical with AoV dsRNA 1 previously reported from A. ochraceus ATCC 28706, which putatively encoded an RNA-dependent RNA polymerase of an unidentified mycovirus. AoR2 was found to encode a putative viral capsid protein (CP) with 63% similarity to that of Penicillium stoloniferum virus S, which was detected from P. stoloniferum ATCC 14586. The function of AoR3 was unknown. The three segments were found to contain a conserved sequence at their 5' termini, while an identical sequence was only found at the 3' termini of AoR1 and AoR2. It is suggested that AoR1, AoR2, and AoR3 originate from an independent partitivirus infecting A. ochraceus. The novel virus is suggested to be Aspergillus ochraceus virus 1, AoV1.

Partitivirus Structure Reveals a 120-Subunit, Helix-Rich Capsid with Distinctive Surface Arches Formed by Quasisymmetric Coat-Protein Dimers

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses.

Intergeneric relationship between the Aspergillus ochraceous virus F and the Penicillium stoloniferum virus S

It was reported that the ‘slow’ component (PsV-S) of Penicillium stoloniferum virus complex also occurred in a second genus, Aspergillus ochraceous. The responsible virus for this intergeneric occurrence was considered to be the ‘fast’ component (AoV-F) of A. ochraceous virus complex. In this investigation, AoV dsRNA 1, that was previously shown to cross-hybridize with PsV-S dsRNA, has been cloned. It was 1754 bp in length and contained one open reading frame of 539 amino acids (p63), had the same genome organization as PsV-S dsRNA S1 and also had the conserved sequence motif of the PsV-S dsRNAs (5′-GCGCAAAA-3′) at the 5′ terminus. A BLAST search indicated that p63 was a putative dsRNA-dependent RNA polymerase (RdRp), had 81% of sequence homology to members of the genus Partitivirus, and grouped together with PsV-S in phylogenetic analysis. But immunoblot analysis showed that the capsid protein (P3) of AoV-F virus component did not reacted against PsV-S antiserum. These evidences suggest that the cross serological relationship between AoV-F and PsV-S previously observed may have been due to the RdRps of the respective viruses rather than between their respective capsid proteins as was assumed in 1985.

Determination of the 5′- and 3′-Terminal Sequences Completes the Sequences of the Two Double-stranded RNAs of Penicillium stoloniferum Virus S

The two genomic segments of Penicillium Stoloniferum virus S (PsV-S), a member of the Partitiviridae, were recently sequenced and published. We independantly sequenced PsV-S and showed that the original sequence was missing nucleotides at both the 5' and 3' termini of both segments. We determined the correct sequence in three independent experiments and found the segments to be 1753 bp (encoding the RNA-dependant RNA polymerase) and 1581 bp (encoding the Capsid Protein). Homology was shown between the 5' and 3' ends of PsV-S and other members of the Partitiviridae.

Genome Organization and Expression of the Penicillium stoloniferum Virus F

The complete sequences of three double-stranded (ds) RNAs (referred to F1, F2 and F3) of Penicillium stoloniferum virus F (PsV-F) were established. The F1 dsRNA was 1677 bp in length, and it contained one open reading frame (ORF) of 538 amino acids (molecular weight of 63 kDa, referred to P63), The F2 dsRNA was 1500 by in length, and also it contained one ORF of 420 amino acids (molecular weight of 46 kDa, referred to P46). The F3 dsRNA was 677 bp in length, but contained a small ORF with unknown function. A sequence motif of (5'-CGTAAAA-3') was found only at the 5' termini of the F1 and F2 dsRNAs, and a sequence motif of (5'-TAAAAAAAAA-3') was found at the 3' termini of all three dsRNA segments. The predicted amino acid sequence of F1 showed 38-48% sequence homology with the putative dsRNA-dependent RNA polymerases (RdRp) of dsRNA viruses, but the predicted amino acid of F2 showed no homology. Phylogenetic analysis using the RdRp sequences of the various Partitiviruses and Alphacryptoviruses revealed that PsV-F clustered well with Partitiviruses, but showed remote relationship with PsV-S. Near full-length and positive-sense single-stranded (ss) RNAs derived from the Fl, F2 and F3 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P63 and P46 showed a positive reaction against PsV-F antiserum, indicating P63 and P46 as RdRp and capsid protein, respectively. These results suggest that PsV-F can be a member of Partitivirus, but it is quite distinct from PsV-S electrophoretically, serologically and genetically, though both viruses coexist in the same cell.

Genome organization and expression of the Penicillium stoloniferum virus S.

The complete sequences of two double-stranded RNAs (dsRNAs) (referred to S1 and S2) of Penicillium stoloniferum virus S (PsV-S) were established. The S1 dsRNA was 1,690 bp in length, and it contained a unique open reading frame (ORF) of 539 amino acids (molecular weight of 62 kDa, referred to P62). The S2 dsRNA was 1,523 bp in length, and also it contained one ORF of 434 amino acids (molecular weight of 47 kDa, referred to P47). Both S1 and S2 ORFs were identified only on the positive strand of each dsRNA segment. A sequence motif of (5'-CUG-3') was found at the 3'-termini of the positive strands of PsV-S1 and S2 dsRNAs. The predicted amino acid sequences of S1 dsRNA showed high sequence homology with the putative RNA-dependent RNA polymerases of RNA viruses. Near full-length and positive-sense single-stranded RNAs derived from the S1 and S2 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P62 and P47 showed a positive reaction against PsV-S antiserum in Western blot analysis. Phylogenetic analysis using the RDRP sequences and the capsid proteins of the various partitiviruses revealed that PsV-S is a definite member of the partitivirus, the family Partitiviridae, and especially clusters well along with D. destructiva virus 1 and 2.

Intergeneric Occurrence of Related Fungal Viruses: the Aspergillus ochraceous Virus Complex and Its Relationship to the Penicillium stoloniferum Virus S

SUMMARY Mycoviruses from Aspergillus ochraceous (ATCC 28706) (AoV) and Penicillium stoloniferum (ATCC 14586) (PsV) were isometric, had diameters of 30 to 32 nm and 32 to 34 nm respectively, and resolved into six bands in CsCl at buoyant densities of 1.275 to 1.357 and 1.285 to 1.390 g/cm3, respectively. All properties of PsV were the same as previous reports except an additional RNA segment with a mol. wt. of 0.51 × 106 and a reverse in the ratio of the major and minor capsid polypeptides. The AoV complex contained nine segments of dsRNA with mol. wt. ranging from 1.12 × 106 to 0.38 × 106, and five thermomelting transition points between 76 and 88°C. It contained capsid polypeptides with mol. wt. of 60000, 51000 and 41000. One dsRNA segment (mol. wt. 1.10 × 106) and one capsid polypeptide (mol. wt. 51000) were common to AoV and PsV-S. Serological tests and virus electrophoresis indicated that at least two serologically distinct viruses were present in A. ochraceous. In double-diffusion serological tests one serological component of AoV and PsV reacted with antiserum of the other virus complex, indicating a serological relationship. A 32P-5′-end-labelled probe of AoV RNA hybridized with PsV RNA and a labelled PsV probe hybridized with AoV RNA. It was concluded that one viral component of the AoV complex is related to the PsV-S component of the PsV complex.

Transcriptase activity assoclated with a type 2 double-stranded RNA mycovirus

It is shown that the virion-associated RNA polymerase of Phialophora virus A, a type 2 double-stranded RNA mycovirus with a genome consisting of three RNA species, is a transcriptase. Synthesis of single stranded RNA in vitro continues for a least 24 hours and after this time two full length transcripts are produced, on average, per dsRNA molecule, i.e.re-initiation of transcription occurs in this in vitro system. Analysis of the products by polyacrylamide gel electrophoresis indicated that the efficiences of transcription of all three double-stranded RNA species were similar. The transcriptase activity of Phialophora virus A differs from the replicase activity of Penicillium stoloniferum virus S, the only other type 2 double-stranded RNA mycovirus whose RNA polymerase has been characterised.

Semi-conservative replication of double-stranded RNA by a virion-associated RNA polymerase

5-Bromo-UTP was found to replace UTP efficiently as a substrate for the virion-associated double-stranded RNA replicase of Penicillium stoloniferum virus PsV-S. The double-stranded RNA product of the replication reaction with 5-bromo-UTP as a substrate gave in equilibrium caesium sulphate density gradient centrifugation a single band with a buoyant density of 1.647 g/ml, consistent with that of a hybrid double-stranded RNA consisting of one brominated and one unbrominated strand. After the reaction none of the original unbrominated double-stranded RNA (buoyant density 1.606 g/ml) could be detected. It is concluded that replication of double-stranded RNA in virions of PsV-S takes place by a semi-conservative mechanism.

Transmission of PsV-F and PsV-S Mycoviruses during Conidiogenesis of Penicillium stoloniferum

SUMMARY: Levels of both fast and slow Penicillium stoloniferum virus (PsV-F and PsV-S) were evaluated in single conidial isolates of P. stoloniferum NRRL5267. Approximately 7 % (3/43) of a random population of conidia from a culture of P. stoloniferum contained no PsV-F, while other conidia produced cultures with various levels of PsV-F. The greatest variation in PsV-F levels was observed in single conidial isolates from cultures that contained low to intermediate levels of virus, i.e. 11 to 25 E 1cm 260 units per g dry weight of mycelium. The least variation was seen in isolates from a culture with a high PsV-F level (37 E 260 units per g dry weight of mycelium). Two of the original 43 single conidial isolates contained no detectable PsV-F or PsV-S. Of the second-generation single conidial isolates from an original PsV-S+ PsV-F- isolate, 6 % failed to show detectable PsV-S. PsV-F levels of cultures remained constant throughout a series of transfers when the inoculum was a mixture of conidia and mycelium. The presence of PsV-F at different levels, or its complete absence, did not affect fungal biomass, conidium size, morphology or viability. The results are discussed in terms of how conidiogenesis may influence the transmission or persistence of PsV-F and PsV-S during morphogenesis.

Initiation complex formation with double stranded RNA

Binding of formylmethionyl tRNA and ribosomes to double stranded RNA has been obtained under conditions identical to those required for initiation complex formation with single stranded RNA. While natural double stranded RNAs from Penicillium chrysogenum virus and Penicillium stoloniferum virus were efficient in forming initiation complexes, the synthetic polynucleotide poly(I).poly (C) was inactive. This suggests that ribosomes can recognize initiation sequences even if these are present in base-paired form.

Action of nucleases on double-stranded RNA.

Double-stranded RNAs from Penicillium chrysogenum virus have been treated with RNAse III, pancreatic RNAse A and RNAse T1 and the degradation of the RNAs has been studied under different conditions. It was found that only the two former enzymes cut across both strands, RNase T1 cannot cleave double strands. RNase III was shown to digest double-stranded RNA by a two step process: an initial phase of specific cleavage is followed by random degradation. In the first phase the enzyme exhibited a definite preference for some specific base pattern. Partial or complete degradation with pancreatic RNase A could also be achieved in media with high salt concentration provided that the enzyme: substrate ratio was increased together with the salt concentration. By combining different assay techniques, the process of degradation was followed from the early stages to complete digestion and the breakdown products were characterised. It is suggested that a structural change in the enzyme molecules enables them to act on double-stranded RNA. RNAse T1, being unable to cleave double strands, provides a useful tool for studying the secondary structure of RNA molecules. Treatment with different nucleases yielded some new information on the structure of different RNA species in Penicillium stoloniferum virus.

Penicillium stoloniferum virus: altered replication in ultraviolet-derived mutants.

Phenotypic mutants of the wild type of Penicillium stoloniferum NRRL5267 were obtained from conidia exposed to ultraviolet light for 60 min (10% survival). Virus content of the wild type and of nine phenotypic mutants was determined by polyacrylamide gel electrophoresis. Four mutants had no detectable Penicillium stoloniferum virus F (PsV-F), whereas the other five had levels of PsV-F in the mycelium similar to the wild-type strain. All nine mutants and the wild type had comparable levels of Penicillium stoloniferum virus S (PsV-S). Maximum virus levels occurred after 9 days of submerged culture in a 2% yeast extract-15% sucrose medium. Virus replication in the fungal host continued after protein, RNA and DNA synthesis levelled off. Virus levels ranged from 85 to 150 E-260 units (extinction units at 260 nm in I cm cell) per 4-7 to 5-3 g dry weight of mycelium for the mutant strains compared to 106 E-260 units per 4-2 g dry weight of the wild-type strain.