Location: Molecular Plant Pathology LaboratoryTitle: RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein Author
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/13/2013
Publication Date: N/A
Technical Abstract: Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between specific sequences and structural elements of CP and RNAs. VLPs are known to encapsidate cellular RNAs, with the level of each RNA species depending on the overall amount of that RNA in the RNA pool. Maize Rayado fino virus (MRFV) CPs can self-assemble in Escherichia coli bacterial cells and Nicotiana benthamiana plants into VLPs packaging cellular RNAs, CP-RNA and mRNA. The RNA encapsidated is often determined by the copies of RNAs in the cellular RNA pool and the type of MRFV coat protein expressed (CP1 or CP2). Here we describe the ability of plant-produced CP1-MRFV-VLPs and CP2-MRFV-VLPs to package different host cellular RNAs as well as foreign reporter genes (GFP). Two methods were used to express MRFV VLPs in N. benthamiana plants: transient expression from the Potato virus X-based vector pP2C2S and agroinfiltration using the pGD vector. VLP encapsidation of RNAs was determined by IC-RNA extraction followed by RNA analysis on denaturing 1% agarose-formaldehyde gels and RT-PCR. Expression from the PVX vector produced MRFV VLPs which encapsidated high amounts of RNA. However, only a portion of this RNA was MRFV-CP gene-specific, the rest being cellular or PVX in origin, suggesting that highly represented foreign RNAs may drive MRFV-VLP assembly toward programmed packaging. To establish if highly representative RNAs in the cellular pool were preferentially encapsidated into VLPs, we co-expressed MRFV, CP1, CP2 and GFP using agroinfiltration in N. benthamiana plants by mixing the bacterial suspension in different ratios to overproduce GFP. MRFV VLPs when MRFV-CP1 was co-expressed with GFP encapsidated GFP RNAs and CP RNAs, unlike VLPs produced by CP2 co-expression with GFP. The latter were found to be empty or contain small RNAs. These results suggest that neither specific nucleotide sequences of the MRFV genome or CP gene are required to form VLPs; however, the 37 amino acid N-terminal extension in-CP1, as well as the overall species concentration of RNA in the cellular pool, may determine the abundance and species of the RNAs packaged.