Synthesis of Foot-and-mouth Disease Virus Capsid Proteins in Insect Cells Using Baculovirus Expression vectors

Abstract

Foot-and-mouth disease virus (FMDV) cDNA cassettes containing sequences encoding the capsid precursor P1-2A with and without those encoding the proteases L and 3C were introduced into Autographa californica nuclear polyhedrosis virus (AcMNPV) expression vectors. Procapsid proteins lAB, 1C and 1D were produced in cells infected with recombinant baculoviruses, when L and 3C were present in the constructs, indicating that these FMDV proteases were active in insect cells. Unlike P1 processing in poliovirus, which has been shown to be catalysed mainly by the 3CD gene product, the 3C protease of FMDV was able to process P 1 independently of 3D. Cytotoxicity of the L protease for insect cells prevented the use of the optimized transfer vector, pAcRP23, for inserting Lcontaining cassettes into AcMNPV. By contrast, viable AcMNPV-FMDV recombinants could be made without restriction on choice of the transfer vector when the L gene was either not expressed or inactivated by an inframe deletion. In the latter case, normal cleavage at the L-P1 junction no longer occurred in cis, and a new processing event, probably catalysed by 3C, was observed within the C-terminal region of the residual L protein. Analysis of baculovirus-expressed products in sucrose gradients showed that a fraction of the capsid proteins is present in an aggregated form, migrating at 70S and possibly resembling FMDV empty capsid particles.

Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy

“…The 2A system does not work in E. coli, 6 but it works well in eukaryotic cell culture from organisms as diverse as insects (Spodoptera frugiperda) 21 or mammalian cell lines (HTK-143, 11 MDBK, 12 BHK-21 13 ). In this work we have demonstrated the correct functioning of the 2A system in other mammalian cells (⌿CRE, ⌿CRIP, NIH/3T3), including human glioma cell line Hs 683 ( Figure 6).…”

Foot-and-mouth disease virus: a long known virus, but a current threat

“…Immunisation of pigs with P1 produced in E. coli resulted in the induction of protection [111]. However, the expression of P1 polypeptide in vaccinia and baculovirus have yielded limited amounts of empty viral capsids [1,138,192,207], whose production requires adequate P1 processing provided by 3C [110,138]. Therefore, further work is required to improve the efficiency of empty capsid formation.…”

Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene

“…This polyprotein system has been used for coexpression of two genes in a variety of eukaryotic systems (e.g. Roosien et al, 1990;Ryan and Drew, 1994;Suzuki et al, 2000;de Felipe et al, 2003), and preliminary work suggests that it can be used to express reporter genes in the cytoplasm of plant cells (Halpin et al, 1999). However, for most applications, this has limited value as many proteins need to be directed to specific subcellular locations in order to fulfill their metabolic functions.…”