Submitted to: Virus Research
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/10/2008
Publication Date: 1/1/2009
Citation: S. Sivakumar, S., Wang, Z., Harrison, R.L., Liu, S., Miller, W.A., Bonning, B.C. 2009. Baculovirus-expressed virus-like particles of Pea enation mosaic virus vary in size and encapsidate baculovirus mRNAs. Virus Research. 139:54-63. Interpretive Summary: Certain virus diseases of plants are spread by insects that feed upon those plants. These plant viruses can be difficult to study, in either the plant or in the insects that transmit the viruses. Using the tools of molecular biology we were able to actually incorporate parts of the plant virus into an insect virus, which could then be more easily studied using cell cultures. The results of this research will be useful to other scientists that are interested in studying and controlling virus diseases of plants.
Technical Abstract: Pea enation mosaic virus (PEMV: family Luteoviridae) is transmitted in a persistent, circulative manner by aphids. We inserted cDNAs encoding the structural proteins of PEMV, the coat protein (CP) and coat protein-read through domain (CPRT) into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus with and without a histidine tag or an upstream Kozak consensus sequence. The Sf21 cell line provided the highest level of CP expression of the cell lines tested and resulted in production of virus-like particles (VLPs). The CPRT was not detected in recombinant baculovirus-infected cells by western blot. Addition of a Kozak sequence increased the yield of baculovirus produced VLPs. Baculovirus expressed VLPs purified on a nickel column were of variable size (13 to 30 nm) and contained CP mRNA. The purified VLPs were also shown by RT-PCR to contain 70% of 154 baculovirus mRNAs, indicative of non-specific RNA encapsidation in the absence of RNA replication. When fed to the pea aphid, Acyrthosiphon pisum (Harris), the VLPs entered the aphid hemocoel confirming that CPRT is not required for uptake of PEMV from the aphid gut. Baculovirus expression of PEMV VLPs provides a useful tool for future analysis of RNA encapsidation requirements, and aphid-virus molecular interactions.