Evolutionary selection of pestivirus variants with altered or no miRNA dependency

Host microRNA (miRNA) dependency is a hallmark of the human pathogen hepatitis C virus (HCV) and was recently also described for the related pestiviruses, which are important livestock pathogens. HCV critically depends on the liver-specific miR-122, whereas the broadly expressed let-7 and miR-17 families bind two sites (S1 and S2) in the pestiviral 3' untranslated region (UTR). Here, we elucidated the miRNA dependency of bovine viral diarrhea virus (BVDV) and confirmed its critical dependence on miR-17 and Argonaute 2 (AGO2). Let-7 binding to S1 had only minor effect on virus production but was required for full translational efficiency. Evolutionary selection experiments using seed site randomized genomes revealed that tropism could be re-directed to different miRNAs. AGO cross-linking and immunoprecipitation (AGO-CLIP) and miRNA antagonism demonstrated that these alternative variants bound and depended on the corresponding miRNAs. Furthermore, we identified variants with miRNA independent replication through acquisition of compensatory mutations near the genomic 3' terminus. Rescue experiments using miRNA mimics demonstrated that miRNA binding and 3' mutagenesis contribute to replication through mutually exclusive mechanisms. Our findings illustrate that pestiviruses, although capable of miRNA independent replication, took advantage of miRNAs as essential host factors, suggesting a favorable path during evolutionary adaptation. Overall design: To study miRNA interactions with bovine viral diarrhea virus (BVDV), cross-linking immunoprecipitation (CLIP) was performed on the Argonatue (AGO) protein. To unambiguously identify interactions with specific miRNAs, we used the CLEAR-CLIP (Moore 2015 Nat Comm) iteration to enrich for miRNA-target chimeras. Prior to cross-linking, bovine Madin-Darby Bovine Kidney (MDBK) cells were infected with wild-type or mutant virus as indicated. Processed reads were aligned to the host (BosTau7) and the viral genome (BVDV). Alignment statistics of CLIP reads are given in the processed data file “Table S1”. A complete table of standard AGO-CLIP reads aligning to BVDV is given in “Table S2”. miRNA-target chimeras were identified in MDBK cells from CLEAR-CLIP experiments as indicated. A complete list of individual miRNA-target chimeras on BVDV is given in “Table S3”.