GCD10 and GCD14 Proteins Facilitate Tomato Bushy Stunt Viral (TBSV) RNA Replication in Saccharomyces Cerevisiae

Tomato bushy stunt virus (TBSV) is a positive-sense RNA virus and its genome replication requires two viral proteins, p33 and RNA-dependent RNA polymerase p92. Previous studies showed that the 5 untranslated region (5UTR) of the genome is important, albeit not essential, for viral RNA replication in plants. Here, my study shows that the 5UTR is also important for viral RNA replication in S. cerevisiae, a model host for TBSV. Using the TBSV 5UTR as bait RNA and a streptotag-based RNA pull-down assay, GCD10 and GCD14 were identified as specific 5UTR-binding proteins. Subsequent electrophoretic mobility shift assay (EMSA) shows that GCD10 and GCD14 proteins bind directly to the 5 UTR and both subunits are required for the binding. To investigate the possible importance of GCD10 and GCD14 proteins in TBSV RNA replication, the replication of a TBSV RNA replicon was analyzed in wild type, GCD10 knockdown and GCD14 knockout yeast strains. Reduction of GCD10 protein and deletion of GCD14 protein in the yeast strains significantly inhibited TBSV subviral defective interfering (DI) RNA replication, suggesting that both GCD10 and GCD14 proteins facilitate TBSV DI replication. Importantly, through western analyses, we confirmed that the reduced levels of the replicon in the yeast strains were not due to altered p33 and p93 protein levels. To gain mechanistic insights on TBSV RNA replication, 5UTR-lacking DI (5'UTR-DI) was also tested in the wildtype, GCD10 knockdown and GCD14 knockout strains. Since 5'UTR-DI replicates without the 5 UTR, its replication was expected to be independent of GCD10 knockdown or GCD14 knockout. As expected, replication of the 5UTR-DI was not inhibited by GCD10 knockdown, showing that GCD10 protein functions only through the 5 UTR. However, replication of 5UTR-DI was inhibited in GCD14 knockout strain, suggesting that GCD14 protein functions through the 5 UTR, but is also able to affect replication by a different mechanism. Although the functions of the 5 UTR binding proteins appear to be complex, my results show that GCD10 and GCD14 proteins are important for TBSV RNA replication in S. cerevisiae. Mechanistic perspectives of the functions of these two proteins are discussed