Coxsackie B3 virus protein 2B contains cationic amphipathic helix that is required for viral RNA replication.
Enterovirus protein 2B has been shown to increase plasma membrane permeability. We have identified a conserved putative amphipathic alpha-helix with a narrow hydrophilic face and an arrangement of cationic residues that is typical for the so-called lytic polypeptides. To examine the functional and structural roles of this putative amphipathic alpha-helix, we have constructed nine coxsackie B3 virus mutants by site-directed mutagenesis of an infectious cDNA clone. Six mutants contained substitutions of the charged residues in the hydrophilic face of the alpha-helix. Three mutants contained insertions of leucine residues between the charged residues, causing a disturbance of the amphipathic character of the alpha-helix. The effect of the mutations on virus viability was assayed by transfection of cells with copy RNA transcripts. The effect on positive-strand RNA replication was examined by introduction of the mutations in a subgenomic luciferase replicon and analysis of luciferase accumulation following the transfection of BGM cells with RNA transcripts. It is shown that both the amphipathy of the domain and the presence of cationic residues in the hydrophilic face of the alpha-helix are required for virus growth. Mutations that disturbed either one of these features caused defects in viral RNA synthesis. In vitro translation reactions and the analysis of viral protein synthesis in vivo demonstrated that the mutations did not affect synthesis and processing of the viral polyprotein. These results suggest that a cationic amphipathic alpha-helix is a major determinant for a function of protein 2B, and possibly its precursor 2BC, in viral RNA synthesis. The potential role of the amphipathic alpha-helix in the permeabilization of cellular membranes is discussed.
van Kuppeveld
F J
FJ
Department of Medical Microbiology, University of Nijmegen, The Netherlands. KUNAZAG3@CAOS.KUN.NL
Galama
J M
JM
Zoll
J
J
van den Hurk
P J
PJ
Melchers
W J
WJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D029822:Enterovirus B, Human / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000096:biosynthesis*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry*
D012367:RNA, Viral; D014764:Viral Proteins