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. 2012 Feb 9;8(2):e1002491. doi: 10.1371/journal.ppat.1002491

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Lin et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Split ubiquitin MYTH assay was used to test binding between the yeast cyclophilins and NoV protein A (panel A), TMV 130K (panel B) and TCV p28 (panel C) replication proteins. The bait proteins were co-expressed with the prey cyclophilin proteins in yeast. The empty prey vector (NubG) was used as a negative control. To launch NoV (panel D) or FHV RNA1 (panel E) replication, we expressed NoV RNA1 from the copper-inducible CUP1 promoter and FHV RNA1 and DI634 from the CUP1 promoter in the parental (BY4741) and in cpr6Δ, and cpr7Δ single deletion and cpr6Δ cpr7Δ double deletion yeast strains. The yeast cells were cultured for 48 hours at 29°C in 3 ml SC-H⁻ with 2% glucose media containing 50 mM CuSO4 (for NoV) and for 24 hours at 29°C in 3 ml SC-H⁻ with 2% galactose (for FHV). Northern blot analysis was used to detect RNA1/RNA3 accumulation for NoV and FHV. The accumulation level of NoV and FHV RNAs was normalized based on 18S rRNA. Each experiment was repeated.