Mycovirus cryphonectria hypovirus 1 elements cofractionate with trans-Golgi network membranes of the fungal host Cryphonectria parasitica

Abstract

The mycovirus cryphonectria hypovirus 1 (CHV1) causes proliferation of vesicles in its host, Cryphonectria parasitica, the causal agent of chestnut blight. These vesicles have previously been shown to contain both CHV1 genomic double-stranded RNA (dsRNA) and RNA polymerase activity. To determine the cellular origins of these virus-induced membrane structures, we compared the fractionation of several cellular and viral markers. Results showed that viral dsRNA, helicase, polymerase, and protease p29 copurify with C. parasitica trans-Golgi network (TGN) markers, suggesting that the virus utilizes the fungal TGN for replication. We also show that the CHV1 protease p29 associates with vesicle membranes and is resistant to treatments that would release peripheral membrane proteins. Thus, p29 behaves as an integral membrane protein of the vesicular fraction derived from the fungal TGN. Protease p29 was also found to be fully susceptible to proteolytic digestion in the absence of detergent and, thus, is wholly or predominantly on the cytoplasmic face of the vesicles. Fractionation analysis of p29 deletion variants showed that sequences in the C terminal of p29 mediate membrane association. In particular, the C-terminal portion of the protein (Met-135-Gly-248) is sufficient for membrane association and is enough to direct p29 to the TGN vesicles in the absence of other viral elements.

FIG. 1.

Fractions of uninfected (EP67) and CHV1-infected (EP802) strains of C. parasitica, obtained by differential centrifugation. Cell lysates were centrifuged at 5,000 × g, 20,000 × g, and 90,000 × g. Pellets (P) and supernatants (S) were collected and analyzed. The microsomal fraction, P90,000, was further fractionated on a Ficoll-²H₂O gradient. The fractions from EP802 that contained viral elements were pooled together and labeled as VF. Corresponding fractions from strain EP67 were also pooled and labeled as such. (A) Western blots of fractions using viral polymerase and helicase antibodies. (B) Nucleic acid extraction from the fractions, with the top panel showing ethidium bromide staining of dsRNA. The bottom panel shows Northern hybridization of the same samples probed with radioactively labeled CHV1-ORF B. (C) Western blots of the same fractions using β-COP and AP-1μ subunit antibodies. Equal total protein amounts were loaded.

FIG. 2.

(A) Western blot of the microsomal fractions of uninfected (EP67) and CHV1-infected (EP802) cells using CHV1-encoded p29 antibodies. (B) Western blot showing subcellular fractionation of cell lysates from CHV1-infected strain EP802 separated by differential centrifugation. Lysates were centrifuged at 5,000 × g, 20,000 × g, and 90,000 × g. Pellets (P) and supernatants (S) were collected and treated with p29, KDEL, Kex2, or GAPDH antibodies. Equal total protein amounts were loaded.

FIG. 3.

Ficoll-²H₂O gradient fractions of the subcellular microsomal fraction, P90,000, of CHV1-infected strain EP802. (A) ▵, Kex2 activity expressed as pmol 7-amino-4-methylcoumarin (AMC) released; •, activity expressed as % Ficoll. (B) Nucleic acid extraction of 100 μl from each gradient fraction and staining with ethidium bromide. (C) Western blot analysis of gradient fractions using antibodies to KDEL, p29, and GAPDH. Fractions were labeled 1 to 11, from top to bottom. For Western assays, equal protein amounts were loaded for all fractions except fraction 1, which did not have enough protein and so the maximal volume was loaded. The percent Ficoll was determined by refractometry.

FIG. 4.

Agarose gels showing Ficoll-²H₂O gradient fractions of the CHV1-infected strain EP802 microsomal fraction, P90,000. The presence of CHV1 dsRNA, p29, CHV1-helicase, KDEL (ER marker), Kex2 (TGN marker), β-COP (intermediate compartment, ER to Golgi marker) and AP-1μ (TGN and endosome marker) in the various fractions were detected using ethidium bromide staining for nucleic acids (dsRNA) and Western blot assays using antibodies to the proteins. The profile of GAPDH is not shown since it was below the detection limit in the gradient fractions.

FIG. 5.

(A) CHV1-p29 hydrophobicity plot. Hydrophobicity was calculated by using the algorithm of Kyte-Doolittle with a window size of 7 amino acids using the SDSC Biology workbench web-based tool (34). (B) Extraction and Western blot analysis of p29 in CHVI-infected strain EP802. The P90,000 fraction was extracted with either 1 M NaCl, 0.1 M Na₂CO₃ (pH 11.5), or 2 M urea and subjected to centrifugation at 90,000 × g, yielding the soluble fraction (S) and pellet (P). The total P90,000 fraction was also fractionated into aqueous (AP) and detergent-soluble (DP) phases after treatment with 1% Triton X-114. Samples were analyzed by Western blotting using p29 antibodies.

FIG. 6.

Protease susceptibility of CHV1 p29. Aliquots of the P90,000 microsomal fraction of CHV1-infected strain EP802 were incubated with increasing amounts of proteinase K. Assays were carried out in the absence (top panel) or presence (bottom panel) of 0.1% Triton X-100. Western blots were generated using p29 antibodies and as a control using antibodies against Kex2. Kex2 is a lumenal (arrow) TGN resident that has a transmembrane domain and a cytoplasmic tail.

FIG. 7.

(A) Schematic representation of p29 deletion constructs. Deletion constructs of p29 coding sequences were fused to GFP. (B) Western blots using p29 antibodies are shown for the microsomal (P90,000) and soluble (S90,000) fractions for each of the deletions. All analyses were done using three independent transformants; representative results are shown.

FIG. 8.

Western blot analysis showing gradient fractions for each of the deletion constructs transformed into strain EP67 and CHV1-infected strain EP802 analyzed by Western blotting using p29 antibodies. Deletion construct nomenclature is the same as for Fig. 7, and gradient fractions are the same as in Fig. 3 and 4. Analyses were carried out using three independent transformants for each construct; representative results are shown.