Publication Type:Journal Article
Source:Virus Res, Volume 139, Number 1, p.54-63 (2009)
ISBN:0168-1702 (Print)<br/>0168-1702 (Linking)
Keywords:Animals, Aphids/metabolism/virology, Baculoviridae/genetics/ metabolism, Cell Line, Cell Nucleus/virology, Gene Expression Regulation, Viral, Luteoviridae/ physiology, Peas/virology, Recombination, Genetic, RNA, Messenger/ metabolism, RNA, Viral/analysis/ metabolism, Spodoptera/metabolism/virology, Viral Structural Proteins/isolation & purification/metabolism, Virus Assembly
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 NTA column were of variable size (13-30 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 viral RNA replication. When fed to the pea aphid, Acyrthosiphon pisum (Harris), the VLPs entered the aphid hemocoel, demonstrating 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 molecular aphid-virus interactions.
Sivakumar, S<br/>Wang, Zhaohui<br/>Harrison, Robert L<br/>Liu, Sijun<br/>Miller, W Allen<br/>Bonning, Bryony C<br/>Research Support, Non-U.S. Gov't<br/>Research Support, U.S. Gov't, Non-P.H.S.<br/>Netherlands<br/>Virus Res. 2009 Jan;139(1):54-63. doi: 10.1016/j.virusres.2008.10.002. Epub 2008 Dec 2.