FP25K and multiplicity of infection impact Autographa californica multiple nucleopolyhedrovirus polyhedra production and gene expression.

摘要

Lepidopteran caterpillars damage important crops such as corn, soybean, and Douglas fir trees. Unfortunately they have become increasingly resistant to chemical insecticides. Baculovirus are a naturally occurring virus that can kill these caterpillars. This virus family has two distinct forms, a budded virus (BV) form to infect cells in culture and a polyhedrin encapsulated form (also known as polyhedra) to infect caterpillars. Continuous cell culture polyhedra production using the BV is not stable and the number of polyhedra containing virus rapidly declines. This decrease is due to mutations in the baculovirus genome with a loss of FP25K protein production. Normally the FP25K is produced by the virus at approximately 24 hpi and assists polyhedra production.; This dissertation concentrates on the FP25K protein and its impact on polyhedra and virus production. Because of the native baculovirus instability alternative locations for expression were investigated. The baculovirus fp25k gene and immediate early promoter (IE1) were used to modify Sf-9 cells to produce low levels of protein constitutively that could briefly increase on infection. The late baculovirus p10 promoter and fp25k gene were used to modify the baculovirus to produce the protein at 24 hpi. When tested, these systems did not produce high numbers of polyhedra with virus. An IE1 cell was developed as a control for the IE1-FP25K cells and during experimentation both were compared to unmodified Sf-9 cells. A low multiplicity of infection (MOI) study was completed that showed the IE1-FP25K cells could produce polyhedra and multiple bundled viruses where other cell lines could not. Unfortunately these polyhedra contained very few viruses. Expression times for early and late mRNAs were then determined for MOIs of 0.1 and 10. The IE1-FP25K cells infected with either MOI could produce mRNA earlier than the other cells. These results prompted the investigation of different promoters to produce higher levels of FP25K protein, development of a specific antibody and characterization of an fp25k gene knock out virus. This work provides a foundation to develop of a stable polyhedra production process that will reduce the use of chemical insecticides.