Contribution of protein p40 to hypovirus-mediated modulation of fungal host phenotype and viral RNA accumulation

Abstract

The papain-like protease p29, derived from the N-terminal portion of the hypovirus CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, was previously shown to contribute to reduced pigmentation and sporulation by the infected host, the chestnut blight fungus Cryphonectria parasitica, while being dispensable for virus replication and attenuation of fungal virulence (hypovirulence). We now report that deletion of the C-terminal portion of p69, which encodes the highly basic protein p40, resulted in replication-competent mutant viruses that were, however, significantly reduced in RNA accumulation. While the Delta p40 mutants retained the ability to confer hypovirulence, Delta p40-infected fungal strains produced more asexual spores than strains infected with either wild-type CHV1-EP713 or a Delta p29 mutant virus. As observed for Delta p29-infected colonies, pigment production was significantly increased in Delta p40-infected fungal strains relative to that in CHV1-EP713-infected strains. Virus-mediated suppression of laccase production was not affected by p40 deletion. A gain-of-function analysis was employed to map the p40 symptom determinant to the N-terminal domain, encompassing p69 amino acid residues Thr(288) to Arg(312). Evidence that the gain of function was due to the encoded protein rather than the corresponding RNA sequence element was provided by introducing frameshift mutations on either side of the activity determinant domain. Moreover, restoration of symptoms correlated with increased accumulation of viral RNA. These results suggest that p40 indirectly contributes to virus-mediated suppression of fungal pigmentation and conidiation by providing an accessory function in hypovirus RNA amplification. A possible role for p40 in facilitating ORF B expression and the relationship between hypovirus RNA accumulation and symptom expression are discussed.

FIG. 1.

Diagram showing the organization of ORF A mutant viruses. The genetic organization of hypovirus CHV1-EP713 is shown at the top. The plus-sense strand of CHV1-EP713 hypovirus dsRNA is 12,712 nucleotides in length, excluding a poly(A) tract, and contains a 495-nucleotide (nt) 5′ noncoding leader sequence, two contiguous ORFs (1,869-nucleotide ORF A and 9,498-nucleotide ORF B) and an 851-nucleotide 3′ noncoding region. ORF A encodes a 69-kDa polyprotein (p69) that is autocatalytically processed into p29 and p40 by the action of a papain-like cysteine protease domain located within the p29 coding region. Key restriction enzyme recognition sites within the full-length CHV1-EP713 infectious cDNA clone, pLDST (9), used for mutant viral cDNA constructions included AflII (CHV1-EP713 map position 450 [28]), BamHI (CHV1-EP713 map positions 562 and 1219), PstI (CHV1-EP713 map position 1846), NcoI (CHV1-EP713 map position 3263), NheI (CHV1-EP713 map position 3705), SpeI (vector sequence), and XbaI (vector sequence). ORF A deletion mutant viruses are shown below the wild-type virus cDNA. Δp40a and Δp40b lack 96.5 and 99.5% of the p40 coding sequence within the context of di- and monocistronic genome organizations, respectively. For Δp40a, two BglII-derived amino acid residues (aspartic acid and proline) were added during the cloning procedure downstream of p40 Arg(2) and fused with p40 Val(612), while the added proline is fused with Met(1) of p48 encoded by ORF B in Δp40b. Δp29, described earlier (11), has a deletion of 87.5% of the p29 gene while retaining the N-terminal 24 codons necessary for virus viability (32). Extreme mutants Δp69a and Δp69b contained 94.4 and 96.1% deletions of the ORF A sequence, again, within the context of the di- and monocistronic genome organizations described for Δp40a and Δp40b, respectively. p29 residue Pro(24) is fused with p40 Val(612) for Δp69a or with p48 Met(1) for Δp69b. A series of nine mutants containing progressive extensions from p40 Val(612) towards the p40 N terminus Leu(254) are presented between Δp69a and Δp29. These extension mutant viruses were used in transfection (gain-of-function) assays to map the region of the p40 coding domain responsible for suppression of pustule formation.

FIG. 2.

(A) Agarose gel electrophoretic analysis of total RNA isolated from C. parasitica colonies transfected with mutant recombinant viruses. Equal amounts (OD, 0.25) of total RNA extracted from uninfected mycelia (lane 7) and mycelia infected with CHV1-EP713 wild-type virus (lane 1) or mutant viruses Δp29 (lane 2), Δp40a (lane 3), Δp40b (lane 4), Δp69a (lane 5), and Δp69b (lane 6) were electrophoresed through a 0.7% agarose gel in the TAE buffer system (40 mM Tris-acetate-1 mM EDTA, pH 7.8) and stained with ethidium bromide. Lane M was loaded in parallel with 1-kb ladder DNA size markers (Gibco-BRL, Gaithersburg, Md.). Relative mobilities of viral dsRNA are indicated. (B) Relative accumulation of viral minus-strand RNA in fungal colonies infected with wild-type CHV1-EP713 and mutant viruses. Total RNA was isolated from mycelia infected with CHV1-EP713 (bar 1) and mutant viruses Δp29 (bar 2), Δp40a (bar 3), Δp40b (bar 4), Δp69a (bar 5), and Δp69b (bar 6) and used for strand-specific cDNA synthesis after denaturation in 90% dimethyl sulfoxide at 65°C. The resulting cDNA was subjected to semiquantitative PCR analysis in a GeneAmp 5700 sequence detection system by using cDNA of 18S rRNA generated in the same RT reaction for normalization. The sequences of primers and TaqMan probes used in the quantification are described in Methods and Materials. Viral minus-strand RNA accumulation levels are reported as percentages of the value for CHV1-EP713-infected colonies, with standard deviations based on three independent measurements indicated by the error bars.

FIG. 3.

Phenotype of C. parasitica colonies infected with mutant viruses. Spheroplasts of C. parasitica strain EP155 were transfected with synthetic transcripts derived from each of the mutant virus cDNA plasmids, Δp29 (3), Δp40a (4), Δp40b (5), Δp69a (6), and Δp69b (7). Virus-free host strain EP155 (1) and wild-type virus (CHV1-EP713)-infected EP155 (2) are also shown. (A) Fungal colonies were photographed after culturing on PDA plates for 6 days. (B) One-month-old cultures of the same colonies were magnified to clearly show formation of conidia-containing pustules.

FIG. 4.

Morphology of cankers induced on dormant chestnut stems by fungal colonies infected with mutant viruses. Dormant chestnut stems were inoculated with freshly grown fungal mycelia transfected with wild-type CHV1-EP713 (pLDST) or mutant viruses Δp29, Δp40a, Δp40b, Δp69a, and Δp69b and incubated in an aquarium under appropriate moisture and temperature conditions for 1 month. Virus-free C. parasitica strain EP155 was also used as an inoculum in parallel. A representative canker photograph is shown for each of the fungal strains.

FIG. 5.

Agarose gel electrophoresis pattern of p40 gain-of-function mutant viral dsRNA. Equal amounts (OD, 0.25) of enriched dsRNA fractions obtained from C. parasitica transfectants with each of the mutant viruses Δp29, p69aΔ25-253, p69aΔ25-271, p69aΔ25-287, p69aΔ25-299, p69aΔ25-312, p69aΔ25-325, p69aΔ25-360, p69aΔ25-384, p69aΔ25-545, and Δp69a were applied to the wells. Electrophoresis was performed in a 0.7% agarose gel in 1× TAE (40 mM Tris-acetate-1 mM EDTA, pH 7.8) as for Fig. 2. Migration positions of 1-kb ladder DNA size markers (lane M) (Gibco-BRL) and mutant viral dsRNA are shown. The dsRNA fractions isolated from Δp29 and p69aΔ25-253 transfectants also contain defective viral dsRNAs that migrate faster than the viral genome. These defective dsRNAs have been generated in many of the transformants examined in this study. However, they are most often observed upon continued passage of the infected mycelium and in the transfectants that accumulate the most dsRNA, e.g., wild-type and Δp29 transfectants. The presence of defective dsRNAs, first described by Shapira et al. (29), does not correlate with any alterations in host phenotype.

FIG. 6.

Enlargements of photographs of C. parasitica colonies infected with p69 extension mutant viruses. Colonies of a healthy C. parasitica EP155 colony and fungal colonies transfected with Δp29 or Δp69 extension mutant viruses p69aΔ25-253, p69aΔ25-271, p69aΔ25-287, p69aΔ25-299, p69aΔ25-312, p69aΔ25-325, p69aΔ25-360, p69aΔ25-384, p69aΔ25-545, and Δp69a were grown on PDA for 1 month at bench top and photographed.

FIG. 7.

ClampR analysis of dsRNA of virus mutants. ClampR was performed on dsRNA recovered from the fungal colonies transfected with mutant virus Δp29, Δp40a, Δp40b, Δp69a, Δp69b, p69aΔ25-253, p69aΔ25-271, p69aΔ25-287, p69aΔ25-299, p69aΔ25-312, p69aΔ25-325, p69aΔ25-360, p69aΔ25-384, and p69Δ25-545. The fragments covering the deletion sites were amplified for all transfectants with a single primer set consisting of NS7 and NS22 (see Table 1 for primer sequences). Amplified fragments were electrophoresed in a 2.0% agarose gel in TBE (89 mM Tris-89 mM boric acid-2.5 mM EDTA, pH 8.3). Lanes M contain 100-bp ladder DNA size standards (Gibco-BRL). Sequence results of ClampR fragments cloned into pCRScript are summarized in Table 6.