title: IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.
abstract: For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens.
authors.affiliation: ICAR-Indian Veterinary Research Institute, Hebbal, Bangalore 560 024, India.
authors.forename: V M
authors.initials: VM
authors.lastname: Vivek Srinivas
authors.affiliation: ICAR-Indian Veterinary Research Institute, Hebbal, Bangalore 560 024, India.
authors.forename: Suresh H
authors.initials: SH
authors.lastname: Basagoudanavar
authors.affiliation: ICAR-Indian Veterinary Research Institute, Hebbal, Bangalore 560 024, India. Electronic address: madhu.hosa@gmail.com.
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Hosamani
mesh_terms: D000086042:3C Viral Proteases; D000818:Animals; D016367:Baculoviridae; D002213:Capsid / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D029544:Foot-and-Mouth Disease Virus; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D000067556:Internal Ribosome Entry Sites; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000067556:Internal Ribosome Entry Sites; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
keywords: Baculovirus expression; Capsid; FMD virus; IRES; Polyprotein processing