This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deng, F.
Right arrow Articles by Nuss, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deng, F.
Right arrow Articles by Nuss, D. L.

 Previous Article  |  Next Article 

Journal of Virology, July 2008, p. 6369-6378, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.02638-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Hypovirus Papain-Like Protease p48 Is Required for Initiation but Not for Maintenance of Virus RNA Propagation in the Chestnut Blight Fungus Cryphonectria parasitica{triangledown}

Fuyou Deng and Donald L. Nuss*

Center for Biosystems Research, University of Maryland Biotechnology Institute, Shady Grove Campus, 9600 Gudelsky Drive, Rockville, Maryland 20850

Received 12 December 2007/ Accepted 23 April 2008


arrow
ABSTRACT
 
The prototypic hypovirus CHV1-EP713, responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, encodes two papain-like proteases, p29 and p48. Protein p29 has been shown to be dispensable for hypovirus RNA replication and to act as a suppressor of RNA silencing. Here we describe a role for p48 in hypovirus RNA propagation. CHV1-EP713 infectious cDNA clones in which the p48 coding region was deleted, {Delta}p48, were unable to establish infection in C. parasitica when introduced as a DNA form by transformation or as a coding strand transcript by electroporation. However, the {Delta}p48 mutant virus RNA was rescued when p48 was provided in trans. Surprisingly, the {Delta}p48 mutant viruses retained replication competence in the apparent absence of p48 following transmission to wild-type C. parasitica and successive subculturing. The replicating {Delta}p48 mutant virus was reduced in RNA accumulation by 60% both in the absence and presence of p48 provided in trans and was transmitted through asexual spores (conidia) at a rate 3 to 8% of that for full-length CHV1-EP713. Complementary analysis of strains expressing p48 or containing the replicating {Delta}p48 mutant virus showed that like p29, p48 contributes to virus-mediated suppression of host pigmentation and conidiation, although to a lesser extent, and is dispensable for hypovirus-mediated hypovirulence. The combined results suggest that papain-like protease p48 plays an essential role in the initiation but not the maintenance of virus RNA propagation and also contributes to the regulation of viral RNA accumulation and vertical transmission.


arrow
INTRODUCTION
 
The literature contains a growing number of examples of correlations between mycovirus infection of plant-pathogenic fungi and reduced fungal virulence (hypovirulence) (reviewed recently in reference 16). While evidence for mycovirus-mediated modulation of fungal virulence is indirect for most mycovirus-fungus interactions, definitive evidence that members of the RNA virus family Hypoviridae (hypoviruses) cause hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, has resulted from the development of a reverse genetics system for several hypoviruses (2, 3, 4, 14). These advances, coupled with a robust DNA-mediated transformation protocol for the haploid C. parasitica (18), provide an experimental system with the unique ability to manipulate the genomes of both a mycovirus and its fungal host (reviewed in reference 10) and to engineer hypoviruses for enhanced biological control potential (reviewed in references 10 and 16).

Hypovirus infection of C. parasitica is persistent and can cause a stable set of phenotypic modifications, such as reduced virulence and reduced sexual and asexual reproduction, but does not cause obvious cytopathic effect or cell death (reviewed in references 15 and 16). While the hypovirus coding strand RNA is infectious when artificially introduced into C. parasitica spheroplasts (2), as for mycoviruses in general, the hypovirus life cycle lacks an extracellular phase. In this regard, hypoviruses do not encode a coat protein or form discrete virus particles (21). Hypoviral RNA genetic information is found as coding strand RNA and as double-stranded RNA associated with membrane vesicles where viral RNA replication proceeds (11, 26). Hypovirus transmission occurs during cytoplasmic mixing as a result of anastomosis (fusion of hyphae) between compatible C. parasitica strains or through asexual spores (conidia) (reviewed in reference 15).

Hypoviruses employ a gene expression strategy that involves the autocatalytic processing of the N-terminal portion of encoded polyproteins by papain-like protease domains. The 12.7-kb RNA coding strand of the prototypic hypovirus CHV1-EP713, encodes two contiguous open reading frames (ORFs) designated ORF A (622 codons) and ORF B (3,165 codons), which contain N-terminal proteases p29 and p48, respectively (7, 21). Protease p29 has been shown to have a surprising number of functions in addition to the cotranslational autocatalytic cleavage of the ORF A polyprotein, p69, at Gly-248 and Gly-249 to liberate itself and the C-terminal domain, p40 (6). Transgenic expression of p29 in C. parasitica in the absence of virus infection results in several symptoms observed for CHV1-EP713-infected strains, primarily reduced orange pigment production, suppressed asexual spore production (sporulation), and reduced extracellular laccase production (5, 9). Deletion of p29 in the context of the CHV1-EP713 infectious cDNA clone, {Delta}p29, partially alleviated these symptoms (9). Although the {Delta}p29 mutant was replication competent, viral RNA accumulation and transmission through conidia were reduced (25). Replication and transmission of the {Delta}p29 deletion mutant were enhanced by expression of p29 either from the fungal chromosome (in trans) or from the CHV1-EP713 genome (in cis) (25). Sun et al. (23) recently reported that p29 augmented the transmission and replication of an unrelated reovirus, MyRV1-Cp9B21. In this regard, Segers et al. (20) reported that p29 acts as a suppressor of RNA silencing both in C. parasitica and in a heterologous plant system. Similarities between p29 and the potyvirus suppressor of RNA silencing HC-Pro have previously been noted (6, 13, 20).

The p48 protease, located at the N terminus of ORF B, resembles p29 in terms of conserved amino acid sequences surrounding the catalytic cysteine and histidine residues and the spacing of these residues relative to the cleavage sites (22). These similarities, the locations of the two proteases at the N termini of the two CHV1-EP713-encoded polyproteins, and sequence alignment studies (13) are consistent with the proposal that the two respective coding regions are products of a gene duplication event (22). We have extended analysis of p48 and now report that like p29, p48 also contributes to CHV1-EP713 mediated suppression of host pigmentation and conidiation, but to a lesser extent than p29. Unlike p29, p48 is not dispensable for viral RNA propagation. However, p48 can rescue the propagation deficiency when supplied in trans. Once rescued, the {Delta}p48 mutant virus RNA can continue to replicate in the apparent absence of p48. Thus, p48 also has multiple functions, including an essential role in the initiation but not the maintenance of hypovirus RNA propagation.


arrow
MATERIALS AND METHODS
 
Construction of p48 deletion virus cDNA and transformation plasmids. Deletion of the p48 coding domain within the context of the CHV1-EP713 infectious cDNA clone, plasmid pLDST (4), was facilitated by use of the pTNR-based mutation modification cassette system as described by Suzuki et al. (24) for construction of CHV1-EP713 gene expression vector candidates. Plasmid pTNR2 contains the CHV1-EP713 5'-terminal 3.7-kb XbaI-NheI fragment derived from pLDST in the background of modified plasmid pTZ19R (U.S. Biochemicals, Cleveland, OH). The p48 deletion mutant, {Delta}p48, was constructed by first replacing the PstI-NheI fragment in pTNR2 (spanning map positions 1 through 3735) with a modified PstI-NheI fragment lacking p48 by PCR amplification from the pLDST plasmid template using Super Taq Plus (Ambion, Austin, TX) and the following two primer sets. Primers FD-F1 (5'-GGTCCACAAAATAAAATTTTAATGGCTGAAGAAGGTTCAGTCTCTG-3') and RS65 (5'-CAATGGGATGACCGGATTGAAC-3') were used to generate a 0.7-kb fragment that extended from the p48 C-terminal cleavage site (nucleotide [nt] 3618) (21) to a position (nt 4325) 3' of the NheI site. Primer FD-F1 also contained a 5', 24-nt extension that corresponded to map positions 2343 to 2366, just 5' of, and including, the p48 translation initiation codon, 2364 to 2366. Primers FD-R1 (CATTAAAATTTTATTTTGTGGACCTTCG-3') and NS42 (5'-CCACAACGCCTTGCTGGTCTCTCC-3') were used to amplify a second 0.7-kb fragment that extended from a position (nt 1644) upstream of the PstI site through the p48 initiation codon (nt 2366). The two fragments were stitched together by a second round of PCR to create the p48 deletion fragment. The {Delta}p48 viral cDNAs were reconstituted by insertion of the 3'-terminal 8.9-kb NheI-SpeI CHV1-EP713 fragment derived from pLDST into the mutated pTNR2.

To construct {Delta}p48 and complementary wild-type viral transformation plasmids containing the benomyl resistance gene marker, the {Delta}p48 and full-length viral cDNAs were released by digesting {Delta}p48 and pLDST plasmids with SspI and SpeI. The cohesive ends were filled in with T4 DNA polymerase (New England Biolabs, Ipswich, MA), and the viral cDNAs were ligated into the HpaI site of transformation plasmid pCPX-NBn1 (20). Plasmid pCPX-NBn1 contains the benomyl resistance cassette derived from Neurospora crassa (18) and an expression cassette consisting of the C. parasitica glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) promoter and terminator regions separated by a polylinker containing the NotI, HpaI, HindIII, and SphI restriction sites, all in a pGEM4Z (Promega, Madison, WI) background. The resulting plasmids were named pXB9 and pXB9-dp48 and differed only in the absence of the p48 coding region in pXB9-dp48. The full-length CHV1-EP713 cDNA in the hygromycin phosphotransferase-containing foundation plasmid pXH9 was also replaced by the mutant {Delta}p48 cDNA to form transformation plasmid pXH9-dp48.

Plasmid p48T used for production of p48-expressing C. parasitica strains was constructed in the foundation transformation vector pCPXHY-1 (9), which confers hygromycin resistance. The p48 coding domain was amplified from cDNA of full-length wild-type hypovirus CHV1-EP713 (in plasmid pLDST) with a primer set consisting of p48F (5'-GACGTCACGGTACCATGTATAAGGAAGCCGAACGACC-3') and p48R (5'-CAGACTGACGCATGCTTATCCAACGAGGATGTCAGGCTC-3'); the sequences in boldface indicate KpnI and SphI recognition sites. The digested fragment was inserted into the KpnI-SphI sites of pCPXHY1 between the GPD promoter and terminator regions to obtain p48T. Plasmid p48TB was constructed from the benomyl resistance-conferring transformation vector pCPX-NBn1. The p48 coding domain was released from p48T with KpnI and SphI, the cohesive ends were filled in with T4 DNA polymerase, and the fragment was ligated into the HpaI recognition site of pCPXNBn1. The orientation of the insertion was confirmed by enzymatic digestion.

Transforming DNA plasmids were introduced by the polyethylene glycol-mediated technique into C. parasitica spheroplasts according to the method of Churchill et al. (8) under conditions described by Choi and Nuss (5). Homokaryons of transformants were obtained through single-conidium germinate selection with corresponding antibiotics. Transcripts generated in vitro from transfection plasmids were electroporated into C. parasitica spheroplasts, using a Gene Pulser II system electroporator (Bio-Rad Laboratories, Hercules, CA) as described by Chen et al. (2).

Viral RNA extraction and quantification. Cultures used for RNA isolation were grown on potato dextrose agar (PDA) overlaid with cellophane for 7 days. Harvested mycelia were frozen in liquid nitrogen and ground into a fine powder with a mortar and pestle. The powder was resuspended in RNA extraction buffer (200 mM NaCl, 100 mM Tris-Cl [pH 8.0], 4 mM EDTA [pH 8.0], 2% sodium dodecyl sulfate, 2 mM dithiothreitol), and total nucleic acid was extracted sequentially with phenol, phenol-chloroform, and chloroform-isoamyl alcohol and precipitated with ethanol. The extracted total nucleic acid was treated with 2 U of RX1 DNase (Promega) in 0.5 ml of 20 mM Tris (pH 8.0)-20 mM MgCl2 in the presence of 40 U of RNasin (Promega) for 1 h at 37 C. Following phenol-chloroform and chloroform extractions, the RNA was precipitated with ethanol and resuspended in 100 µl double-distilled water. The relative levels of viral RNA accumulation in fungal colonies infected with wild-type and p48 deletion virus were measured by real-time reverse transcriptase PCR (RT-PCR) as described by Suzuki and Nuss (26), but using random priming in the RT reaction to measure accumulation of both positive- and negative-strand viral RNA. Nonquantitative agarose gel (1%) analysis of hypovirus double-stranded RNA (dsRNA) was performed on RNA samples enriched in dsRNA by ethanol precipitation of the 2 M lithium chloride soluble fraction.

Phenotypic measurements. Differences in colony morphology for hypovirus-free C. parasitica strain EP155, CHV1-EP713- and mutant virus-infected strains, and CHV1-EP713 viral gene transformants were examined following incubation on PDA plates for 1 week on the laboratory benchtop at 22 to 24°C and a light intensity of approximately 1,300 lx. Sporulation was measured from cultures grown on PDA for 20 days. Conidia were washed from the plates with 0.015% Tween 80 and counted with a hemacytometer. Virulence assays were performed with dormant American chestnut tree stems as previously described (12), with six duplicate inoculations per fungal strain. Inoculated stems were kept at room temperature in a glass tank to maintain moisture. Cankers were measured 1, 2, and 3 weeks after inoculation. Statistical analysis for virulence and sporulation was performed using the Proc GLM procedure of SAS version 8.0 (SAS Institute, Cary, NC), and the type I error rate ({alpha}) was set at 0.05.

Virus transmission assay. Fungal colonies were cultured on PDA for 3 weeks on a lab benchtop with a 12-h photoperiod. Conidia were harvested, diluted, and spread onto PDA plates. The plates were cultured for 2 to 3 days to allow spore germination. Single-spore germinates were transferred to new PDA plates and cultured for an additional 2 to 4 weeks in order to score infected germinates. Virus or mutant virus infections were examined for virus-specific phenotypic markers, including reduced pigmentation, reduced sporulation, and altered colony morphology.

Total protein extraction and Western blotting. Total protein extracts for Western blotting were prepared by the method of Parsley et al. (19). Dilution immunoblot quantification of relative p48 protein levels was performed as described previously using rabbit antisera raised against recombinant hypovirus CHV1-EP713 proteins expressed from map position nt 2364 to 4070 (the antigen used to generate antibody B1 included all of the p48 coding region) or from map position nt 6672 to 7674 (the antigen used to generate antibody B7 included a region just upstream of and extending into the CHV1-EP713 RNA-dependent RNA polymerase [RDRP] domain) (21).


arrow
RESULTS
 
Protease p48 is required for viral RNA propagation. Previous studies have shown that deletions of p29, p40, or the entire p69 coding sequence from ORF A, with the exception of the first 24 codons, resulted in replication-competent mutant CHV1-EP713 viruses (9, 24, 26), indicating that these proteins were not necessary for viral replication. It was of interest to test whether deletion of the p48 coding domain would also result in replication-competent mutant virus: i.e., to determine whether p48 is also dispensable for viral RNA propagation. To ask this question, we deleted the p48 coding sequence (Fig. 1A) from the full-length viral cDNA (plasmid pLDST) to make transfection plasmid {Delta}p48 and electroporated transcripts generated from the control pLDST and mutant {Delta}p48 plasmids into spheroplasts of C. parasitica strain EP155. Colonies transfected with full-length viral transcripts exhibited the typical set of phenotypic changes associated with CHV1-EP713 infection (2), and viral dsRNAs were detected in nucleic acid extracts (data not shown). In contrast, colonies transfected with {Delta}p48 deletion transcripts failed to show any infection-associated phenotypic changes and no viral dsRNA was detected in nucleic acid extracts. Thus, transcripts derived from the {Delta}p48 deletion plasmid were unable to establish an infection when introduced into C. parasitica spheroplasts under conditions in which transcripts of full-length CHV1-EP713 readily establish infections.


Figure 1
View larger version (47K):
[in this window]
[in a new window]

  		FIG. 1. (A) Genomic organization of prototypic hypovirus CHV1-EP713. The CHV1-EP713 RNA coding strand is 12,712 nt in length, excluding a poly(A) tract, and contains a 495-nt 5'-noncoding leader sequence, two contiguous ORFs (1,869-nt ORF A and 9,498-nt ORF B), and an 851-nt 3'-noncoding region. ORF A encodes two polypeptides, p29 and p40, that are autocatalytically released from polyprotein p69 by the action of a papain-like cysteine protease domain located within the p29 coding region. ORF B encodes a large polyprotein that contains an N-terminal papain-like protease, p48, and conserved polymerase and helicase domains (modified from reference 15). "An" is the equivalent of poly(A), i.e., the number of A residues. (B) Western blot analysis of protein extracts made from putative p48 transformant strains using antibody B1 (Materials and Methods) to detect p48 protein. Lane C contains protein extract from the control C. parasitica strain EP155, which, in this case, was transformed with plasmid pXH9-dp48, which contains the CHV1-EP713 viral cDNA lacking the p48 coding domain in the foundation plasmid pXH9 (Materials and Methods). The arrow to the left denotes the band that corresponds to p48, and the asterisk denotes a slower-migrating anti-p48 cross-reactive band that is also present in protein extracts of wild-type strain EP155. Putative transformants designated T-4, T-5, T-10, and T-16 in this figure expressed p48 protein at comparable levels and were used in subsequent studies in this report under the designations p48T-4, p48T-5, p48T-10, and p48T-16, respectively. The transformants designated T-2T were taken through two successive transformations with p48T, but were not used further in this study. (C) End-point dilution immunoblot analysis to determine the relative accumulation of p48 in a CHV1-EP713-infected strain and p48T transformant strains. Wild-type strain EP155 served as a negative control. A total of 20 µg of protein extracts (see Materials and Methods) was loaded for wild-type strain EP155 and two p48 transformant strains, p48T-4 and p48T-5, while a series of dilutions of protein extracts, ranging from 1 µg to 0.02 µg, were loaded for the CHV1-EP713-infected EP155 strain. The signal intensities for the p48T-4 and p48T-5 strains were similar to those observed for the 0.1- and 0.02-µg samples of the EP155/CHV1-EP713 extract, indicating a difference of about 400-fold.

The hypovirus cDNA transformation system (4) was also used to examine the role of p48 in CHV1-EP713 RNA propagation. The p48 deletion virus cDNA and full-length CHV1-EP713 cDNA were both cloned into plasmid pCPX-NBn1 to obtain plasmids pXB9-dp48 and pXB9, respectively, which differ only in the absence or presence of the p48 coding region. Similar to the transfection results, colonies transformed with pXB9, containing the full-length CHV1-EP713 cDNA, exhibited the typical CHV1-EP713 infection phenotype and accumulated viral dsRNAs (data not shown). In contrast, colonies transformed with pXB9-dp48 exhibited slightly reduced pigmentation and conidiation due to the expression of p29 from transcripts derived from the integrated viral cDNA, but failed to exhibit the full CHV1-EP713 infection phenotype or to accumulate viral dsRNA. These results further support the conclusion that protease p48 is required for CHV1-EP713 RNA propagation.

Protease p48 can function in trans to complement the p48 deletion virus. In view of the report by Suzuki and coworkers that hypovirus protease p29 could function in trans to enhance viral dsRNA accumulation (25), it was of interest to determine whether p48 could function in trans to complement the replication deficiency of the {Delta}p48 deletion virus. The first approach was to ask whether the {Delta}p48 transcripts could establish an infection when introduced into a transgenic C. parasitica strain that was expressing p48 (p48 transformant). Putative p48 transformants were screened by Western blot analysis to confirm the expression of p48 protein as shown in Fig. 1B. Transformants p48T-4, p48T-5, p48T-10, and p48T-16 expressed p48 at comparable levels and performed similarly in all subsequent studies described in this report. As expected, the level of expression from the nuclear copy of the p48 coding region under the control of the C. parasitica GAPDH gene promoter and terminator was found to be substantially less than the level found in CHV1-EP713 infected cells. Based on end-point dilution immunoblot analysis, it is estimated that p48 is present in CHV1-EP713-infected mycelia at a level approximately 400 times higher than in the p48 transformant strains examined (Fig. 1C).

In contrast to the failure of {Delta}p48 transcripts to establish an infection when introduced into EP155 spheroplasts, the p48 deletion mutant transcripts readily established infections when electroporated into the spheroplasts prepared from p48 transformant strains. Mycelia formed by hypovirus-infected transfected p48 transformant spheroplasts on transfection regeneration medium exhibited a morphology typical of infected C. parasitica, with flat colony margins (Fig. 2; p48T-10/{Delta}p48) rather than the colony margins with aerial hyphae produced by uninfected C. parasitica (Fig. 2, EP155/{Delta}p48), and accumulated viral dsRNA (Fig. 3). The dsRNA species isolated from the p48 deletion virus-infected p48 transformants migrated faster than the RNA isolated from CHV1-EP713-infected strains (Fig. 3; p48T-10/{Delta}p48 and p48T-16/{Delta}p48 dsRNA in lanes 2 and 3). Reverse transcription-PCR using primers flanking the p48 coding domain confirmed this observation; the RT-PCR products generated from dsRNAs isolated from strains p48T-10/{Delta}p48 and p48T-16/{Delta}p48 were about 1.2 kb smaller than the RT-PCR product of full-length virus (Fig. 3, lanes 5 and 6). Nucleotide sequencing further confirmed the deletion of the p48 coding sequences.


Figure 2
View larger version (54K):
[in this window]
[in a new window]

  		FIG. 2. Protease p48 complementation of the replication-defective {Delta}p48 deletion mutant virus. (A) Mycelia of EP155 (left) and p48 transformant p48T-10 (right) transfected with transcripts of the {Delta}p48 mutant virus are shown growing on transfection regeneration medium. The transfected wild-type EP155 strain produced the aerial hyphae generally produced by regenerated uninfected C. parasitica strains, while the transfected p48 transformant strain produced little aerial hyphae symptomatic of hypovirus infection. Two sectors of uninfected aerial hyphae can be observed in the p48T-10/{Delta}p48 regeneration plate at the top right. (B) Delayed development of p48 deletion virus in double-transformation strains (in trans complementation). Transgenic strain p48T-10 transformed with pXH9-dp48 (p48 deletion construct) showed wild-type phenotypes (e.g., which produced pigmentation and conidial spores), when cultured early after transformation, while initial transformant colonies of the wild-type strain EP155 transformed with full-length virus construct (pXB9) showed virus-infected phenotypes, such as reduced growth, pigmentation, and conidiation. (C) Colonies of single-spore isolates of p48T10/pXB9-dp48 transformants develop virus-infected phenotypes after culture for 2 to 5 days on PDA plates. Note that the center of the culture exhibited an uninfected colonymorphology. (D) Two colonies isolated after anastomosis between p48T-10 and EP155/pXB9-dp48 transformants are shown. In each case, delayed development of virus-infected phenotypes was also observed for the colonies recovered from anastomosis plates.


Figure 3
View larger version (64K):
[in this window]
[in a new window]

  		FIG. 3. Agarose gel (1%) analysis of dsRNAs recovered from full-length and p48 deletion virus-infected colonies and reverse transcription PCR confirmation of the p48 deletion of the mutant virus. Lanes 2 and 3 contain the dsRNA-enriched fractions from p48 transgenic strains p48T-10 and p48T-16 transfected with p48 deletion viral transcripts ({Delta}p48), respectively. The {Delta}p48 dsRNAs are smaller than dsRNA recovered from wild-type strain EP155 transfected with full-length viral (pLDST) transcripts (lane 1). The region containing the p48 coding domain was amplified from the isolated dsRNAs by reverse transcription-PCR using primers NS42 (CHV1-EP713; map positions 1644 to 1667) and RS60 (map positions 4080 to 4100). The PCR products of p48 deletion virus (lanes 5 and 6) are 1.2 kb smaller in size than that generated from full-length virus dsRNA (lane 4) (1,202 bp versus 2,456 bp). Nucleotide sequencing of these PCR products further confirmed the p48 deletion. M, molecular size marker.

A second approach for determining the ability of p48 to rescue {Delta}p48 mutant virus replication involved double transformation with the mutant virus cDNA and the p48 coding domain. A very high percentage (>90%) of the transformants picked from regeneration plates following transformation of EP155 spheroplasts with the CHV1-EP713 full-length cDNA (pXB9) exhibited a virus-infected colony morphology following transfer to PDA plates (Fig. 2B), while none of the over 100 transformants recovered after transformation with the {Delta}p48 deletion mutant cDNA (pXB9-dp48) ever showed a virus-infected colony morphology. In contrast, a high percentage of strains doubly transformed with p48T and pXB9-dp48 exhibited a virus-infected colony morphology, but in a delayed fashion. Irrespective of whether transformation plasmid pXB9-dp48 was used to transform a p48 transformant strain or the p48T plasmid was used to transform a pXB9-dp48 transformant strain, the resulting double transformants exhibited a virus-free phenotype and produced abundant pycnidia and conidial spores when initially transferred to PDA (Fig. 2B; p48T-10/pXB9-dp48). However, the majority of these double transformants exhibited the virus-infected phenotype after single-conidium isolation, although the full virus-infected colony morphology took 2 to 5 days to develop (Fig. 2C).

Anastomosis was used as a third test of in trans complementation. This is a process observed for most filamentous fungi in which the hyphae of the same strain or compatible strains fuse (form anastomoses) and exchange cytoplasmic contents. Pairing of a p48 transformant strain and a {Delta}p48 mutant virus transformant strain resulted in the rescue of the {Delta}p48 virus at the site where the two colonies merged and subsequent spread of the replicating mutant virus into newly developing hyphae to give colonies infected with the replicating {Delta}p48 mutant virus, as indicated in Fig. 2D. Similar to the double-transformation test results, delayed development of virus-infected phenotypes was observed. Results of all three tests indicate that p48 can complement the propagation deficiency of p48 deletion virus RNA.

Protease p48 is required for the initiation of infection but not for the maintenance of CHV1-EP713 RNA replication. We next asked whether the {Delta}p48 deletion mutant virus, once rescued by p48 supplied in trans, required p48 for maintenance of replication by anastomosis-mediated transfer of the replicating {Delta}p48 virus from a p48 transformant strains to virus-free C. parasitica strain EP155. Surprisingly, the replicating {Delta}p48 virus was not only transmitted to the wild-type strain but continued to replicate in strain EP155 after successive subculturing that involved transfer of a 2-mm mycelial plug of the culture to new PDA culture plates after 7 to 10 days of incubation. Viral RNA was detected as in Fig. 3 after as many as eight subculturings (data not shown). These {Delta}p48 virus-infected cultures were antibiotic sensitive (i.e., did not contain nuclei from the donor strain). The unlikely possibility of the presence of any residual donor strain nuclei was removed by transmission of the replicating {Delta}p48 mutant virus to colonies derived from germinated uninucleate, asexual spores (conidia) isolated from the infected EP155 recipient strain.

Replication of the p48 deletion virus in wild-type C. parasitica strain EP155 following transfer from p48 transformant strains was further confirmed by Western blot analysis. As shown in Fig. 4, the level of p48 protein in p48 transformant strains (lanes 4 and 5) that contained the replicating {Delta}p48 virus mutant was similar to the level in an uninfected p48 transformant strain (lane 6) and much lower than the accumulation of p48 in the CHV1-EP713-infected strain (lane 1), consistent with results shown in Fig. 1. In contrast, p48 protein was not detected in protein extracts of the single-spore wild-type EP155 strains containing the transmitted replicating {Delta}p48 deletion virus (Fig. 4A, lanes 2 and 3). However, antibody {alpha}B7, raised against amino acid sequences corresponding to viral nucleotide sequences 6672 to 7674, which corresponds to the region just upstream of and extending into the viral RDRP domain, detected viral proteins in protein extracts of all strains containing the replicating CHV1-EP713 (lane 1) and {Delta}p48 deletion virus, including the wild-type EP155 strains (lanes 2 and 3), but not in the uninfected p48T transformant strain (lane 6). We conclude from these combined results that once rescued by p48 supplied in trans, the {Delta}p48 mutant virus can maintain replication in the absence of detectable p48 protein.


Figure 4
View larger version (56K):
[in this window]
[in a new window]

  		FIG. 4. Western blot analysis of viral protein levels in p48 transgenic and virus-infected strains. A large quantity of p48 protein was detected with antibodies directed against p48 ({alpha}B1) in protein extracts isolated from strain EP155 infected with full-length CHV1-EP713 (lane 1), while much smaller quantities of p48 protein were detected in extracts made from p48 transgenic strains (p48T-10; lane 6) and the p48 transgenic strains infected with the p48 deletion virus (two independent p48T-10/{Delta}p48 strains; lanes 4 and 5, respectively). However, p48 protein was undetectable in extracts from wild-type strain EP155 infected with p48 deletion virus (single-spore strains {Delta}p48-S4 and {Delta}p48-S9; lanes 2 and 3, respectively). The arrow denotes the band that corresponds to p48, and the asterisk denotes a slower-migrating anti-p48 cross-reactive band which is also present in protein extract of wild-type strain EP155. (B) Virus ORF B-encoded proteins were detected with an antibody raised against a region from just upstream of and extending into the CHV1-EP713 RDRP domain ({alpha}B7; see Materials and Methods) in extracts of all virus-infected strains, including wild-type strain EP155 infected with p48 deletion virus (lanes 2 and 3). The bands migrating slower than the 175-kDa marker were the major B7 antibody-reacting polypeptides and are currently being characterized. The ORF B-encoded proteins were not detected in extracts made from uninfected p48 transgenic strain p48T-10 (lane 6).

Phenotypic changes caused by transgenic expression of p48 and by the replicating {Delta}p48 mutant virus. Since p29 transformant strains exhibit a number of phenotypic traits that are also observed in CHV1-EP713-infected colonies (9), principally reduced orange pigmentation and reduced condiation, it was of interest to examine p48 transformant strains for phenotypic changes. As indicated in Fig. 5, p48 transformants, represented by p48T-10 and p48T-16, were slightly reduced in pigmentation and the production of conidium-containing pycnidia compared to control strain EP155, but not to the same degree as exhibited by the CHV1-EP713 strain. However, the effect of p48 on pigmentation and condiation was more pronounced in p48 transformant strains that contained multiple copies of the transgenic p48 coding region when cultured for 14 days under a slightly higher light intensity (1,600 lx versus 1,300 lx) (data not shown).


Figure 5
View larger version (115K):
[in this window]
[in a new window]

  		FIG. 5. Effect of p48 transformation on C. parasitica pigmentation and conidiation. The p48 transformant strains, exemplified by strains p48T-10 and p48T-16, exhibit slightly reduced pigmentation and conidiation levels compared to the wild-type strain after incubation at 22 to 24°C under a low light intensity of 1,300 lx for 7 days.

The colony morphology exhibited by C. parasitica strains harboring the replicating {Delta}p48 mutant virus was clearly different from that of CHV1-EP713-infected strains. As shown in Fig. 6, strain EP155 and p48T transformant strains harboring the replicating {Delta}p48 mutant produced colonies with uneven margins and little aerial hyphae that grew more slowly than CHV1-EP713-infected strains. Under standard growth conditions, the {Delta}p48 replicating virus and CHV1-EP713 appeared to have similar effects on host pigmentation and conidiation. However, after incubation for 25 days under increased light intensity (1,600 lx), the p48 deletion viruses produced a small amount of pycnidia and conidia while the wild-type CHV1-EP713-infected strain did not (Fig. 6 and Table 1). This observation provides further support for the view that p48 contributes to hypovirus-mediated reductions of pigmentation and conidiation.


Figure 6
View larger version (130K):
[in this window]
[in a new window]

  		FIG. 6. Colony morphology of wild-type strain EP155, CHV1-infected strain EP713, wild-type strain EP155, and p48 transgenic strain p48T-10 infected with the replicating p48 deletion virus {Delta}p48, EP155/{Delta}p48, and p48T-10/{Delta}p48, respectively. The photograph was taken on day 25 of culture on PDA rather than day 7 as in Fig. 5. Note that the p48 deletion virus-infected colonies had pronounced irregular margins and produced small numbers of pycnidia and conidia, while the full-length virus-infected colonies (EP155/CHV1-EP713) did not. Conidiation results are presented in Table 1 for cultures incubated for 20 days.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Conidiation by C. parasitica strains infected with p48 deletion virus {Delta}p48

Deletion of p48 results in reduced accumulation and vertical transmission of virus genomic RNA. The accumulation of p48 deletion virus RNA in wild-type strain EP155 and p48 transgenic strains was compared to the accumulation of CHV1-EP713 RNA. As quantified by real-time RT-PCR (Fig. 7B), the RNA levels for the {Delta}p48 deletion virus were significantly lower in both the wild-type and p48 transformant strains relative to the level of viral RNA in CHV1-EP713-infected strains. It is also noteworthy that transgenic expression of p48 does not significantly increase the {Delta}p48 mutant virus RNA level.


Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 7. Real-time RT-PCR quantification of {Delta}p48 virus RNA accumulation. Viral RNA accumulation levels were measured as described in Materials and Methods and are reported as percentages of the value obtained for CHV1-EP713-infected colonies. Note that there are no significant differences in mutant {Delta}p48 viral RNA levels in wild-type (EP155/{Delta}p48-S4 and EP155/{Delta}p48-S9) and p48 transgenic strains (p48T-5/{Delta}p48 and p48T-16/{Delta}p48) infected with p48 deletion virus (i.e., in the presence or absence of p48 provided in trans). Strains EP155/{Delta}p48-S4 and EP155/{Delta}p48-S9 are EP155 strains that received {Delta}p48 virus by anastomosis and were subcultured and then single spored.

When isolating single conidial spores for confirmation that {Delta}p48 mutant virus replication was maintained in wild-type strain EP155, we noticed that virus transmission rates appeared low. Therefore, the vertical transmission of the {Delta}p48 deletion virus was further investigated and compared with that of full-length virus and {Delta}p29 mutant viruses. The mean frequency values for virus transmission through conidia are shown in Table 2. The {Delta}p29 deletion virus was transmitted at an efficiency of 46.2%, compared to 100% exhibited by wild-type virus CHV1-EP713, confirming the previously reported results (1, 25). However, the {Delta}p48 deletion virus in p48 transformant and wild-type strains showed further reductions in transmission rate, ranging from 3.0% to 8.6%. Suzuki and coworkers have reported that low levels of virus in infected colonies and conidial spores were correlated with reduced vertical transmission (25, 26). However, in this case, the RNA levels of {Delta}p48 and {Delta}p29 mutant viruses were quite similar (data not shown) while the vertical transmission efficiencies were quite different (3.0 to 8.6% versus 46.2%; Table 2). The low level of {Delta}p48 virus transmission relative to that for the {Delta}p29 mutant virus raises the possibility that p48 may contribute to virus transmission through conidia. However, the fact that similar low levels of {Delta}p48 virus transmission were observed from both infected EP155 and p48 transformant strains suggests that such a role requires p48 to be supplied in cis or to be present at a concentration higher than that found in p48 transformant strains (Fig. 1).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Efficiency of hypovirus transmission through conidia

Protease p48 is dispensable for hypovirus-mediated hypovirulence. The virulence of p48 transgenic and {Delta}p48 deletion virus-infected EP155 strains was tested and compared with wild-type and CHV1-EP713-infected strain EP155. As illustrated in the representative photographs in Fig. 8 and the quantitative data presented in Table 3, the p48 transformant strains produced large cankers similar to those caused by wild-type strain EP155, with densely packed orange spore-containing stromal pustules protruding through the bark surface. In contrast, {Delta}p48 deletion virus-infected strains were severely reduced in the ability to expand on chestnut tissue, forming small cankers that resemble cankers formed by hypovirus CHV1-EP713-infected strain EP155. These results indicate that protease p48 does not contribute directly to CHV1-EP713-mediated hypovirulence.


Figure 8
View larger version (56K):
[in this window]
[in a new window]

  		FIG. 8. Virulence assay on dormant American chestnut stems. Shown are representative cankers formed by wild-type strain EP155, strain EP155 infected with CHV1-EP713 (EP155/CHV1-EP713), p48 transgenic strains p48T-4 and p48T-10, and wild-type strain EP155 infected with p48 deletion virus (EP155/{Delta}p48-S4 and EP155/{Delta}p48-S9). Photographs of cankers were taken 3 weeks postinoculation.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Virulence of C. parasitica strains infected with p48 deletion virus {Delta}p48 or transformed with the p48 coding domain


arrow
DISCUSSION
 
Mechanisms underlying the replication of hypovirus RNAs are poorly understood. The results of this study show that hypovirus-encoded protein p48 is required for viral RNA propagation and can promote viral RNA replication when provided in trans, but is not required for maintenance of viral RNA replication once it has been initiated. We also show that p48 contributes to hypovirus-mediated symptoms, such as reduced pigmentation and reduced conidiation, but does not contribute to reduced fungal virulence.

Like the related p29 protease, p48 appears to serve multiple functions in the infected fungal host, several of which overlap with those observed for p29. The rescued replicating {Delta}p48 mutant virus had less of a suppressive effect on fungal conidiation than wild-type virus (Fig. 6 and Table 2), and transgenic expression of p48 caused a low but consistently observable reduction in fungal pigment production and conidiation (Fig. 5). While the magnitude of the changes in pigmentation and conidiation caused by p48 was less than that caused by p29 (9), the contribution of p48 to virus-mediated symptom expression in CHV1-EP713-infected strains, where it is present at a 400-fold higher concentration (Fig. 1), may be significant.

Both p29 and p48 also contribute to viral RNA accumulation and virus transmission, but with some interesting differences. The {Delta}p29 and {Delta}p48 mutant virus RNAs accumulated to similar levels and approximately 20% the level of CHV1-EP713 viral RNA accumulation. Interestingly, the levels of accumulation of {Delta}p48 virus RNA were the same in wild-type EP155 and in the p48 transformant strains, while the {Delta}p29 viral RNA accumulation increased to near CHV1-EP713 levels when p29 was provided in trans (25). The reduction in {Delta}p29 virus RNA accumulation was previously correlated with a reduction in virus transmission through asexual spores that was increased concomitant with the increase in virus RNA accumulation when p29 was provided. Interestingly, the level of transmission observed for the {Delta}p48 mutant virus was much lower than that observed for the {Delta}p29 virus and was not increased by the supply of p48 in trans. These observations suggest that p48 contributes to transmission through conidia in a manner that is independent of viral RNA accumulation and only when provided in cis. Remarkably, like p29, p48 does not contribute directly to CHV1-EP713-mediated hypovirulence.

Considerations of the potential role that p48 plays in viral RNA replication must be prefaced by a brief discussion of hypovirus taxonomy and an account of the little that is known about hypovirus RNA replication strategy. The initial classification of hypoviruses as dsRNA viruses was based on the observed presence of prominent dsRNA species on agarose gel profiles of RNA extracts isolated from hypovirulent strains of C. parasitica (reviewed in reference 15). The characterization, cloning, and sequence analysis of the dsRNAs isolated from a hypovirulent C. parasitica strain infected with prototypic hypovirus CHV1-EP713 revealed a 12.7-kbp RNA with one strand containing a 3'-poly(A) and the complementary strand containing a 5'-poly(U) (21). All of the coding information was found within the poly(A)-containing strand. Phylogenetic analysisis strongly suggested a common ancestry with the single-stranded positive-sense plant potyviruses (13). However, the absence of a virus-encoded coat protein and resulting absence of discrete virus particles in hypovirus-infected mycelia complicated the assignment of the viral genomic RNA in the conventional sense. Experimental evidence to support the classification of hypoviruses as single-stranded, positive-sense RNA viruses was subsequently provided by the demonstration (2) that the full-length CHV1-EP713 poly(A) coding strand RNA is infectious. A reassessment of the current taxonomic classification of hypoviruses as dsRNA viruses (17) is clearly needed.

Hypovirus RDRP activity has been identified in association with membrane vesicles isolated from hypovirus-infected mycelia (11). The RDRP reaction products consisted primarily of coding strand transcripts and a smaller amount of dsRNA that corresponds to replicative intermediate or replicative form structures (11). Deletion of the p48-related CHV1-EP713 protease p29 resulted in a reduction in hypovius RNA accumulation (26). While dispensable for viral RNA replication, p29 was able to restore viral RNA accumulation to wild-type levels when provided in trans (25). The contribution of p29 to viral RNA accumulation is likely related to its recently identified role as a virus-encoded suppressor of the host antiviral RNA silencing pathway (20). In this regard, the ability of p48 to suppress RNA silencing is under investigation.

In contrast to the result observed for p29 deletion, CHV1-EP713 RNA transcript deleted of p48, {Delta}p48, is replication incompetent. The ability of p48 to rescue the {Delta}p48 deletion viral RNA was demonstrated by three different methods: transfection of the {Delta}p48 transcript into a transformed fungal strain expressing p48 from a nuclear copy (Fig. 2), sequential transformations with the p48 coding domain and the {Delta}p48 mutant virus cDNA (Fig. 2), and anastomosis of a p48-expressing transformed strain with a strain transformed with the {Delta}p48 mutant cDNA (Fig. 2). In the first method, rescue of the {Delta}p48 transcript occurs during the regeneration of transfected spheroplasts, so no difference was observed in the onset of the virus-infected phenotype caused by wild-type and rescued {Delta}p48 virus. Interestingly, a delay in the development of the infection phenotype was always observed for rescue by double transformation or by anastomosis, relative to the onset observed for wild-type CHV1-EP713. One interpretation of this observation is that p48 needs to accumulate to a critical level or ratio relative to the {Delta}p48 mutant viral RNA in order to initiate replication.

Most intriguing is the apparent hit-and-run mechanism by which p48 promotes the initiation of viral RNA propagation when provided in trans, but is then dispensable for subsequent maintenance of viral RNA replication. The rescued {Delta}p48 mutant virus continues to replicate after anastomosis-mediated transfer to wild-type strain EP155, even after successive subculturing that would clearly dilute out any p48 protein that might have been transferred with the {Delta}p48 viral RNA. The EP155 recipient strains supporting {Delta}p48 viral RNA replication showed no evidence of antibiotic resistance due to the presence of a residual population of nuclei from the donor p48 transformant strains. Finally, the replicating {Delta}p48 mutant viral RNA was transmissible to antibiotic-sensitive mycelia germinated from single uninucleate conidia derived from the {Delta}p48-infected EP155 strain. The selection of single conidial isolates of C. parasitica is the standard method for ensuring nuclear homogeneity.

One explanation for these results is that the propagation deficiency of the {Delta}p48 viral RNA is rescued by a second mutation that allows subsequent replication of the {Delta}p48 RNA in the absence of p48. However, the {Delta}p48 transcript, when expressed from a nuclear cDNA copy in {Delta}p48 cDNA-transformed fungal strains, has not been observed to mutate to replication competence in the absence of p48 even after continuous subculturing of multiple independent transformants for extended periods. Consequently, if a second mutation is responsible for the p48-rescued {Delta}p48 virus to subsequently replicate in the absence of p48, that mutation event must be p48 dependent. Efforts to further test this possibility are in progress.

A more likely scenario to explain the hit-and-run mechanism involves a p48-mediated conformational change in the viral RNA or recruitment of host/viral protein replication factors that, once in place, trigger a switch from translation of the viral RNA positive-strand to minus-strand synthesis or the establishment of a functional membrane-associated replication complex. Once minus-strand synthesis is initiated and progresses to an optimal level or the membrane-associated replication complex is operationally established, p48 is no longer required for maintenance of replication, which may then continue by a cis-preferential process. A transient requirement of a virus-encoded protein for viral RNA replication has not been widely described. The p48-related protein interactions and structural requirements involved in this unusual mechanism are currently under investigation.


arrow
ACKNOWLEDGMENTS
 
This work was supported in part by Public Health Service grant GM55981 to D.L.N.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Center for Biosystems Reseach, University of Maryland Biotechnology Institute, Shady Grove Campus, 9600 Gudelsky Drive, Rockville, MD 20850. Phone: (240) 314-6218. Fax: (240) 314-6225. E-mail: nuss{at}umbi.umd.edu Back

{triangledown} Published ahead of print on 30 April 2008. Back


arrow
REFERENCES
 
    1
  1. Chen, B., C. Chen, B. H. Bowman, and D. L. Nuss. 1996. Phenotypic changes associated with wild-type and mutant hypovirus RNA transfection of plant pathogenic fungi phylogenetically related to Cryphonectria parasitica. Phytopathology 86:301-310.[CrossRef]
    2. 2
  2. Chen, B., G. H. Choi, and D. L. Nuss. 1994. Attenuation of fungal virulence by synthetic infectious hypovirus transcripts. Science 264:1762-1764.[Abstract/Free Full Text]
    3. 3
  3. Chen, B., and D. L. Nuss. 1999. Infectious cDNA clone of hypovirus CHV1-Euro7: a comparative virology approach to investigate virus-mediated hypovirulence of the chestnut blight fungus Cryphonectria parasitica. J. Virol. 73:985-992.[Abstract/Free Full Text]
    4. 4
  4. Choi, G. H., and D. L. Nuss. 1992. Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA. Science 257:800-803.[Abstract/Free Full Text]
    5. 5
  5. Choi, G. H., and D. L. Nuss. 1992. A viral gene confers hypovirulence-associated traits to the chestnut blight fungus. EMBO J. 11:473-477.[Medline]
    6. 6
  6. Choi, G. H., D. M. Pawlyk, and D. L. Nuss. 1991. The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro. Virology 183:747-752.[CrossRef][Medline]
    7. 7
  7. Choi, G. H., R. Shapira, and D. L. Nuss. 1991b. Cotranslational autoproteolysis involved in gene expression from a double-stranded RNA genetic element associated with hypovirulence of the chestnut blight fungus. Proc. Natl. Acad. Sci. USA 88:1167-1171.[Abstract/Free Full Text]
    8. 8
  8. Churchill, A. C. L., L. M. Ciuffetti, D. R. Hansen, H. D. Van Etten, and N. K. Van Alfen. 1990. Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids. Curr. Genet. 17:25-31.[CrossRef]
    9. 9
  9. Craven, M. G., D. M. Pawlyk, G. H. Choi, and D. L. Nuss. 1993. Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus. J. Virol. 67:6513-6521.[Abstract/Free Full Text]
    10. 10
  10. Dawe, A. L., and D. L. Nuss. 2001. Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annu. Rev. Genet. 35:1-29.[CrossRef][Medline]
    11. 11
  11. Fahima, T., P. Kazmierczak, D. R. Hansen, P. Pfeiffer, and N. K. Van Alfen. 1993. Membrane-associated replication of an unencapsidated double-stranded RNA of the fungus, Cryphonectria parasitica. Virology 195:81-89.[CrossRef][Medline]
    12. 12
  12. Hillman, B. I., R. Shapira, and D. L. Nuss. 1990. Hypovirulence-associated suppression of host functions in Cryphonectria parasitica can be partially relieved by high light intensity. Phytopathology 80:950-956.[CrossRef]
    13. 13
  13. Koonin, E. V., G. H. Choi, D. L. Nuss, R. Shapira, and J. C. Carrington. 1991. Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses. Proc. Natl. Acad. Sci. USA 88:10647-10651.[Abstract/Free Full Text]
    14. 14
  14. Lin, H., X. Lan, H. Liao, T. B. Parsley, D. L. Nuss, and B. Chen. 2007. Genome sequence, full-length infectious cDNA clone, and mapping of viral double-stranded RNA accumulation determinant of hypovirus CHV1-EP721. J. Virol. 81:1813-1820.[Abstract/Free Full Text]
    15. 15
  15. Nuss, D. L. 1992. Biological control of chestnut blight: an example of virus-mediated attenuation of fungal pathogenesis. Microbiol. Rev. 56:561-576.[Abstract/Free Full Text]
    16. 16
  16. Nuss, D. L. 2005. Hypovirulence: mycoviruses at the fungal-plant interface. Nat. Rev. Microbiol. 3:632-642.[CrossRef][Medline]
    17. 17
  17. Nuss, D. L., B. I. Hillman, D. Rigling, and N. Suzuki. 2005. Family Hypoviridae, p. 597-601. In C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (ed.), Virus taxonomy: VIIIth report of the International Committee on Taxonomy of Viruses. Elsevier, London, United Kingdom.
    18. 18
  18. Orbach, M. J., E. B. Porro, and C. Yanofsky. 1986. Cloning and characterization of the gene for β-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol. Cell. Biol. 6:2452-2461.[Abstract/Free Full Text]
    19. 19
  19. Parsley, T. B., B. Chen, L. M. Geletka, and D. L. Nuss. 2002. Differential modulation of cellular signaling pathways by mild and severe hypovirus strains. Eukaryot. Cell 1:401-413.[Abstract/Free Full Text]
    20. 20
  20. Segers, G. C., R. van Wezel, X. Zhang, Y. Hong, and D. L. Nuss. 2006. Hypovirus papain-like protease p29 suppresses RNA silencing in the natural fungal host and in a heterologous plant system. Eukaryot. Cell 5:896-904.[Abstract/Free Full Text]
    21. 21
  21. Shapira, R., G. H. Choi, and D. L. Nuss. 1991. Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight. EMBO J. 10:731-739.[Medline]
    22. 22
  22. Shapira, R., and D. L. Nuss. 1991. Gene expression by a hypovirulence-associated virus of the chestnut blight fungus involves two papain-like protease activities. J. Biol. Chem. 266:19419-19425.[Abstract/Free Full Text]
    23. 23
  23. Sun, L., D. L. Nuss, and N. Suzuki. 2006. Synergism between a mycoreovirus and a hypovirus mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713. J. Gen. Virol. 87:3703-3714.[Abstract/Free Full Text]
    24. 24
  24. Suzuki, N., L. M. Geletka, and D. L. Nuss. 2000. Essential and dispensable virus-encoded replication elements revealed by efforts to develop hypoviruses as gene expression vectors. J. Virol. 74:7568-7577.[Abstract/Free Full Text]
    25. 25
  25. Suzuki, N., K. Maruyama, M. Moriyama, and D. L. Nuss. 2003. Hypovirus papain-like protease p29 functions in trans to enhance viral double-stranded RNA accumulation and vertical transmission. J. Virol. 77:11697-11707.[Abstract/Free Full Text]
    26. 26
  26. Suzuki, N., and D. L. Nuss. 2002. Contribution of protein p40 to hypovirus-mediated modulation of fungal host phenotype and viral RNA accumulation. J. Virol. 76:7747-7759.[Abstract/Free Full Text]

Journal of Virology, July 2008, p. 6369-6378, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.02638-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Chiba, S., Salaipeth, L., Lin, Y.-H., Sasaki, A., Kanematsu, S., Suzuki, N. (2009). A Novel Bipartite Double-Stranded RNA Mycovirus from the White Root Rot Fungus Rosellinia necatrix: Molecular and Biological Characterization, Taxonomic Considerations, and Potential for Biological Control. J. Virol. 83: 12801-12812 [Abstract] [Full Text]  
  • Guo, L.-h., Sun, L., Chiba, S., Araki, H., Suzuki, N. (2009). Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713. Nucleic Acids Res 37: 3645-3659 [Abstract] [Full Text]  
  • Zhang, X., Nuss, D. L. (2008). A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus. Proc. Natl. Acad. Sci. USA 105: 16749-16754 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deng, F.
Right arrow Articles by Nuss, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deng, F.
Right arrow Articles by Nuss, D. L.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»