IDENTIFICATION AND CHARACTERIZATION OF HOST FACTORS INVOLVED IN TOMBUSVIRUS REPLICATION

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI RNA) via plasmids in yeast resulted in robust replication of the viral RNA. Previous work using a yeast single gene deletion library (YKO) revealed 96 yeast genes affecting virus replication. The essential yeast genes could not be deleted so we used the Yeast Tet Promoters Hughes Collection (yTHc) where the original promoter was replaced by Tetracyclin-titratable promoter. I tested the 800 essential host genes available in yTHc. In total, we found 30 new host genes whose down-regulated expression either increased or decreased the accumulation of a TBSV repRNA. The identified essential yeast genes fall into different categories on the basis of the cellular processes they are involved in, such as RNA transcription/metabolism, protein metabolism/transport etc. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts of p33 and p92(pol) viral replication proteins, (ii) the activity of the tombusvirus replicase complex, and (iii) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous YKO screening of yeast led to the identification of 126 host genes (out of ~5,600 genes that represent ~95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA. In the YKO screening, we found NSR1 (homologous to plant nucleolin) gene, whose deletion led to increased TBSV repRNA accumulation. Nucleolin is an abundant RNA binding protein, which shuttles between the nucleolus, the nucleoplasm and the cytoplasm. This protein is involved in rRNA maturation, ribosome assembly and regulation of cellular RNA metabolism.We found that over-expression of Nsr1p in yeast or nucleolin in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Temporal regulation of Nsr1p over-expression revealed that the inhibitory effect of Nsr1p was more profound when it was expressed at early stages of viral replication. In vitro binding experiments showed that Nsr1p binds preferably to the RIII in the repRNA (which is derived from 3’ UTR of viral genome). Consistent with its RIII specific binding, over-expression of Nsr1p only reduced 40% of the accumulation of TBSVΔRIII repRNA in yeast. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the in vitro replication assay. Our data suggest that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication