Overexpression of BMV CP can selectively repress translation from nonreplicating RNA reporters. (A) Schematic representations of reporter RNAs used to examine the effect of CP on translation. The reporters RNAs contained GFP flanked by 5′ and 3′ UTR from the three BMV genomic RNAs. The names of the reporters denote the sources of the 5′ and 3′ UTRs. For example, 1GFP2 will denote that the 5′ UTR is from RNA1 while the 3′ UTR is from RNA2. (B) Fluorescent microscopic examination of GFP expression from RNA reporters in N. benthamiana. Each of culture expressing reporter RNA (at OD595 of 0.2) was coinfiltrated with Agrobacterium expressing either the empty vector or BMV CP (OD595 of 1.5). GFP images were taken at 40 hpi with a filter set at an excitation wavelength of 540 nm and an emission range of 505–530 nm. (C) Western blotting analysis of GFP protein accumulation. The leaves were macerated and approximately equal amounts of protein lysates were loaded into SDS-PAGE and then subjected to Western blot analysis. The identical samples in parallel blots were probed with monoclonal antibody against GFP or rabbit polyclonal serum that recognizes the BMV CP. The image labeled “Rubisco” was from a Coomassie blue-stained Western blot membrane and is intended to serve as a protein loading control. The gel image labeled GFP-mRNA was from an RT-PCR experiment that semiquantitatively analyzed the GFP mRNA level. No bands were observed in the samples that lacked reverse transcription reaction (data not shown). 18S ribosomal RNA was amplified from the same samples as the loading control.
