Proteome-wide overexpression of host proteins for identification of factors affecting tombusvirus RNA replication: an inhibitory role of protein kinase C

Abstract

To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.

Fig 1

Grouping of the identified yeast proteins affecting TBSV replication based on their known cellular functions. Systematic screening of the yeast overexpression library resulted in the identification of 141 unique yeast genes that either promoted or inhibited TBSV replication (Table 1). The identified host genes were grouped into 11 categories based on their known cellular and biochemical functions. The number of genes in each category is shown in parentheses. Proteins with multiple functions were placed arbitrarily only in one of the categories, mainly based on their predicted function in TBSV replication.

Fig 2

Overexpression of Pkc1p inhibits TBSV repRNA accumulation in yeast. (A) Schematic representation of the known domains in the tombusvirus replication protein p33. TMD, transmembrane domain; P, phosphorylation site; RPR, arginine-proline-rich RNA-binding domain. S1 and S2 are subdomains of the p33:p33-p92 interaction domain. The PSVP late domain, in connection with ubiquitinated lysines, is involved in binding to the host ESCRT components. (B) Binding of the unphosphorylated (left) and phosphorylated (right) forms of p33 to the viral RNA (71, 74). Phosphorylation is performed by Pkc in vitro, and the phosphorylated serine and threonine residues in p33 located in the vicinity of the RPR motif are shown in bold and labeled with the letter P. Note that the positively charged arginines within the RPR motif, critical in binding to the viral RNA, are predicted to be neutralized by the phosphorylated serine and threonine, as shown, resulting in a lack of RNA binding by p33. (C) Reduced TBSV repRNA accumulation in yeast overexpressing yeast Pkc1p. Overexpression was done from the GAL1 promoter. repRNA replication took place for 24 h at 29°C before RNA analysis. The accumulation level of DI-72 (+)repRNA (shown as percentages) was normalized based on that of 18S rRNA. Each experiment was repeated three times. (D) To launch TBSV repRNA replication, we expressed His₆-p33 and His₆-p92 from the copper-inducible CUP1 promoter and DI-72 (+)repRNA from the constitutive ADH1 promoter in the parental (wt, BY4741) and pkc1^ts yeast strains. The yeast cells were cultured for 36 h at either 23°C (permissive temperature) or 32°C (semipermissive temperature) on 2% glucose SC minimal medium. Northern blot analysis was used to detect DI-72 (+)repRNA accumulation. The accumulation level of DI-72 (+)repRNA was normalized based on 18S rRNA. Each experiment was repeated three times. (E) Overexpression of yeast Pkc1p in pkc1^ts yeast strains reduced TBSV repRNA accumulation. The yeast cells were cultured for 36 h at 32°C (semipermissive temperature).

Fig 3

Effect of a Pkc1 inhibitor (cercosporamide) on viral RNA accumulation in yeast. (A) (Top) Northern blot analysis was used to detect DI-72 (+)repRNA accumulation in a yeast strain treated with cercosporamide (0.5 μg/ml) to inhibit Pkc1p function. rec, recombinant RNA; deg, partially degraded. (Bottom) Ethidium-bromide stained gel of total RNA extracts of the samples used for Northern blotting above. (B to D) Northern blot analysis of TBSV repRNA replication in yeast expressing the wt p33 or the nonphosphorylatable p33-A₂₀₅A₂₁₀A₂₁₁ (B) or partially phosphorylatable p33-A₂₁₀A₂₁₁ (C) and in p33-D₂₀₅ mutants from the ADH1 promoter (D), while wt p92 and DI-72 repRNA were expressed from the CUP1 promoter and the GAL1 promoter, respectively. Yeast was cultured for 36 h at 23°C in the presence of cercosporamide (1.0 μg/ml), 2% galactose, and 50 μM CuSO₄.

Fig 4

The effect of cercosporamide treatment on TBSV and TCV RNA accumulation in N. benthamiana protoplasts. (A) Northern blot analysis was used to detect genomic (g) TBSV RNA accumulation in protoplasts treated with cercosporamide to inhibit Pkc1-like functions. Protoplasts from N. benthamiana were electroporated with TBSV gRNA and treated with various concentrations of cercosporamide (2- and 4-μg/ml final concentrations). Total RNA samples were obtained 40 h postelectroporation. The ethidium-bromide stained gel at the bottom shows rRNA levels. Note that treatment with ethanol (shown as “0”), which is used to dissolve cercosporamide, was chosen as the control. The accumulation level of TBSV RNA was normalized based on the rRNA. Each experiment was repeated three times. (B) As panel A, except that TCV RNA was used for protoplast electroporation.

Fig 5

The effect of cercosporamide treatment on TBSV RNA accumulation in N. benthamiana leaves. (A) Symptom intensification caused by TBSV infection in plants treated with cercosporamide. Note that leaf curling (the young leaves are circled) is more pronounced in TBSV-infected plants 4 days postinoculation. (B) Northern blot analysis was used to detect gTBSV RNA. Treatment of N. benthamiana leaves with cercosporamide promotes the accumulation of TBSV RNAs. Total RNA samples from the inoculated leaves were obtained 4 days postinoculation and used for Northern blotting (top) and gel analysis (bottom) to show rRNA levels.

Fig 6

Inhibition of TBSV replication by recombinant Pkc1p in vitro. (A) Scheme of the CFE-based TBSV replication assay. Purified recombinant p33 and p92^pol replication proteins of TBSV and in vitro-transcribed TBSV DI-72 (+)repRNA were added to the whole-cell extract prepared from the wt yeast strain. The purified recombinant yeast Pkc1p was added before (exp. C) or during (exp. B) the CFE-based TBSV replication assay. (B) Denaturing PAGE analysis of the ³²P-labeled TBSV repRNA products obtained in the in vitro CFE-based TBSV replication assay in the presence of recombinant Pkc1p (1× = 200 ng). Each experiment was repeated three times. (C) CFE-based assay similar to that in panel B, except that Pkc1p was preincubated with p33/p92 in the reaction buffer for 30 min at 25°C. (D) Western blot analysis of purified recombinant GST-Pkc1p with anti-GST antibody.