Trans-Encapsidation of Flock House virus with Tobacco Mosaic virus Structural Proteins

Abstract

Flock House virus (FHV) encodes a viral polymerase supporting autonomous replication of the FHV genome which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. As FHV viral genome size is strictly limited by native FHV capsid, the goal of this study was to determine if this restriction could be eliminated by inserting a non-native viral packaging signal from Tobacco Mosaic virus (TMV) to allow for trans-encapsidation by TMV coat. FHV was adapted to express enhanced green fluorescent protein (GFP) and several FHV-GFP expressing clones were generated from which full length RNA transcripts were generated. Fluorescent microscopy was used to confirm GFP expression after FHV-GFP RNA transfection into baby hamster kidney (BHK-21) cells. The TMV origin of assembly (OA) was introduced into the most robust GFP expressing FHV construct and GFP expression was used to ensure functional FHV RNA 1 activity. RNA from the best clone, FHV-C2-GFP-OA, was then mixed with TMV coat protein and monitored for encapsidation. The production of TMV-like rod shaped nanoparticles indicated that FHV-GFP-OA RNA can be encapsidated in vitro by purified TMV coat protein. This is the first demonstration of replication-independent but specific encapsidation/packaging of the FHV genome by heterologous plant viral structural proteins while maintaining FHV genome replication capacity.