title: Modification of the trypsin cleavage site of rotavirus VP4 to a furin-sensitive form does not enhance replication efficiency.
abstract: The infectivity of rotavirus (RV) is dependent on an activation process triggered by the proteolytic cleavage of its spike protein VP4. This activation cleavage is performed by exogenous trypsin in the lumen of the intestines in vivo. Here, we report the generation and characterization of a recombinant RV expressing cDNA-derived VP4 with a modified cleavage site (arginine at position 247) recognized by endogenous furin as well as exogenous trypsin. Unexpectedly, the mutant virus (KU//rVP4-R247Furin) was incapable of plaque formation without an exogenous protease, although the mutant VP4s on virions were efficiently cleaved by endogenous furin. Furthermore, KU//rVP4-R247Furin showed impaired infectivity in MA104 and CV-1 cells even in the presence of trypsin compared with the parental virus carrying authentic VP4 (KU//rVP4). Although the total titre of KU//rVP4-R247Furin was comparable to that of KU//rVP4, the extracellular titre of KU//rVP4-R247Furin was markedly lower than its cell-associated titre in comparison with that of KU//rVP4. In contrast, the two viruses showed similar growth in a furin-defective LoVo cell line. These results suggest that intracellular cleavage of VP4 by furin may be disadvantageous for RV infectivity, possibly due to an inefficient virus release process.
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Komoto
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Mitsutaka
authors.initials: M
authors.lastname: Wakuda
authors.affiliation: Laboratory of Electron Microscopy, Fujita Health University Joint Research Laboratory, Toyoake, Aichi 470-1192, Japan.
authors.forename: Tomihiko
authors.initials: T
authors.lastname: Ide
authors.affiliation: Laboratory of Electron Microscopy, Fujita Health University Joint Research Laboratory, Toyoake, Aichi 470-1192, Japan.
authors.forename: Gen
authors.initials: G
authors.lastname: Niimi
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Maeno
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Higo-Moriguchi
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D053837:DNA Cleavage; D045683:Furin / Q000235:genetics* / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D009154:Mutation; D012401:Rotavirus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014357:Trypsin / Q000737:chemistry; D010948:Viral Plaque Assay; D014771:Virion / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; C060339:VP4 protein, Rotavirus; D014357:Trypsin; D045683:Furin