Cyclophilin A binds to the viral RNA and replication 2 proteins resulting in inhibition of tombusviral replicase 3 assembly 4 5 6 7

18 Replication of plus-stranded RNA viruses is greatly affected by numerous host-coded 19 proteins acting as restriction factors. Cyclophilins, which are a large family of cellular 20 prolyl isomerases, have previously been found to inhibit Tomato bushy stunt tombusvirus 21 (TBSV) replication in a yeast model based on genome-wide screens and global proteomics 22 approaches. In this paper, we further characterized single-domain cyclophilins, including 23 the mammalian Cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast 24 Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that 25 recombinant CypA, Roc1 and Roc2 strongly inhibited TBSV replication in a cell-free 26 replication assay. Additional in vitro studies revealed that CypA, Roc1 and Roc2 27 cyclophilins bound to the viral replication proteins and CypA and Roc1 to the viral RNA, 28 too. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral 29 replicase complex and viral RNA synthesis. A catalytically inactive mutant of CypA was 30 also able to inhibit TBSV replication in vitro due to binding to the replication proteins and 31 the viral RNA. Over-expression of CypA and its mutant in yeast or plant leaves led to 32 inhibition of tombusvirus replication, confirming that CypA is a restriction factor for 33 TBSV. Overall, the current work revealed a regulatory role for the cytosolic single-domain 34 Cpr1-like cyclophilins in RNA virus replication. 35 36