title: Triple layered rotavirus VLP production: kinetics of vector replication, mRNA stability and recombinant protein production.
abstract: Rotavirus infection causes diarrhoeal disease in infants, killing more than half million children each year. Virus-like particles (VLP) seem to be excellent vaccine candidates, since they are cheaper to produce than attenuated viral vaccines and safer, as they do not contain genetic material. The present work focus on a triple layered particle composed by three rotavirus structural proteins: VP2, VP6 and VP7, produced in an insect cell/baculovirus expressing system. Two strategies were evaluated for 2/6/7 VLP production: co-infection with three monocistronic baculovirus vectors or single-infection with a tricistronic multi-gene baculovirus vector; these strategies were followed at different levels: baculovirus DNA replication kinetics, mRNA stability, protein production and VLP formation. This study highlights some of the reasons why the tricistronic baculovirus strategy is more efficient for production of triple layered rotavirus 2/6/7 VLP than monocistronic co-infection, in particular: (i) the tricistronic vector presents higher DNA replication rates than the monocistronic vectors, (ii) the mRNA stability is invariant for all mRNAs corresponding to VP2, VP6 and VP7 and (iii) the tricistronic baculovirus strategy produces an excess of VP7 over VP6 when compared to the VP7/VP6 stoichiometric ratio in the native rotavirus. Although the co-infection strategy leads to protein production akin to the rotavirus VP7/VP6 stoichiometric ratio, the tricistronic vector strategy yields higher amounts of rotavirus-like particles.
authors: Instituto de Biologia Experimental e Tecnológica (IBET), Apartado 12, 2781-901 Oeiras, Portugal.
authors: Helena L A
authors: HL
authors: Vieira
authors: Catarina
authors: C
authors: Estêvão
authors: António
authors: A
authors: Roldão
authors: Cristina C
authors: CC
authors: Peixoto
authors: Marcos F Q
authors: MF
authors: Sousa
authors: Pedro E
authors: PE
authors: Cruz
authors: Manuel J T
authors: MJ
authors: Carrondo
authors: Paula M
authors: PM
authors: Alves
mesh_terms: D000818:Animals; D005822:Genetic Vectors / Q000502:physiology*; D042822:Genomic Instability / Q000235:genetics; D007700:Kinetics; D015202:Protein Engineering / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins