Identification of Host Factors Interacting with a γ-Shaped RNA Element from a Plant Virus-Associated Satellite RNA

Abstract

Previously, we identified a highly conserved, γ-shaped RNA element (γRE) from satellite RNAs of cucumber mosaic virus (CMV), and we determined γRE to be structurally required for satRNA survival and the inhibition of CMV replication. It remains unknown how γRE biologically functions. In this work, pull-down assays were used to screen candidates of host factors from Nicotiana benthamiana plants using biotin-labeled γRE as bait. Nine host factors were found to interact specifically with γRE. Then, all of these host factors were down-regulated individually in N. benthamiana plants via tobacco rattle virus-induced gene silencing and tested with infection by GFP-expressing CMV (CMV-gfp) and the isolate T1 of satRNA (sat-T1). Out of nine candidates, three host factors, namely histone H3, GTPase Ran3, and eukaryotic translation initiation factor 4A, were extremely important for infection by CMV-gfp and sat-T1. Moreover, we found that cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 contributed to the replication of CMV and sat-T1, but also negatively regulated CMV 2b activity. Collectively, our work provides essential clues for uncovering the mechanism by which satRNAs inhibit CMV replication.

The non-template functions of helper virus RNAs create optimal replication conditions to enhance the proliferation of satellite RNAs

“…The trans -replication system, a method previously described for studying CMV replication in N . benthamiana plants [ 51 , 59 ], was employed in this study to assess the influence of viral RNAs and their variants on satRNA replication. Briefly, the Agrobacterium cells harboring the plasmids used for transiently expressing the RNA silencing suppressor P19, CMV replication proteins, and one of satRNAs, were mixed with an equal proportion, along with the bacterial cells carrying either an empty vector (pCB301) or a plasmid expressing one of viral RNAs.…”

“…Two μg of total RNAs were separated on a 1.5% agarose gel containing 7% formaldehyde. The separated RNAs were transferred onto a positively changed nylon membrane (GE) and hybridized with the digoxin (DIG)-labeled DNA oligonucleotide probes, specifically designed for detecting CMV genomic RNAs or satRNAs [ 51 , 59 ]. In addition, the oligonucleotide probe used for detecting the LS RNA3 mutants lacking the conserved sequence in the 3′ untranslated region (3′ UTR) is complementary to the sequence positioning from 1299 to 1333 nt of LS RNA3.…”