The non-template functions of helper virus RNAs create optimal replication conditions to enhance the proliferation of satellite RNAs
Fig 5
Complete replication of viral RNAs is required for enhancing satRNA replication in trans-replication assays.
(A) Schematic diagrams of ncL3 and its derivative ncL3-ΔVRΔCRΔ5U. The green blocks denote the substitution of the 5′ UTR with the sequence of M13R(-48), and the orange blocks denote the deletion of both VR and CR in the 3′ UTR. (B) Northern blotting analyses of the accumulation of sat-T1, ncL3 and its derivatives ncL3-ΔVRΔCR and ncL3-ΔVRΔCRΔ5U. In the trans-replication assay expressing LS replication proteins (L1a+L2a), sat-T1 was co-expressed with ncL3 or its derivatives, as well as the vector (pCB301) control in the 5th true leaves of Nicotiana benthamiana plants. At 3 days post-infiltration, total RNAs were extracted from the infiltrated leaves and subjected to northern blot hybridization. The relative accumulation levels of RNA3, and positive-sense or negative sense RNAs of sat-T1 are shown below. Ethidium bromide-stained ribosomal RNAs served as the loading control. (C) Strand-specific RT-PCR for detecting the negative-sense RNAs of ncL3 and its derivative in the RNA samples shown in panel (B). RNA samples were digested with TURBO DNase to remove DNA, and subjected to RT reactions with the primer ncL3a-F. The RT products were amplified using the primer pair ncL3a-F and ncL3b-R. In parallel, total RNAs were amplified directly in PCR reactions without the RT process, indicated with RT(-). PCR products were separated in a 1% agarose gel and observed under UV light after ethidium bromide staining. “M” denotes the DL2000 DNA ladder.