COXSACKIE B3 VIRUS PROTEIN 2B CONTAINS A CATIONIC AMPHIPATHIC HELIX THAT IS REQUIRED FOR VIRAL-RNA REPLICATION

Enterovirus protein 2B has been shown to increase plasma membrane perm eability. We have identified a conserved putative amphipathic alpha-he lix with a narrow hydrophilic face and an arrangement of cationic resi dues 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-di rected mutagenesis of an infectious cDNA clone. Six mutants contained substitutions of the charged residues in the hydrophilic face of the a lpha-helix. Three mutants contained insertions of leucine residues bet ween the charged residues, causing a disturbance of the amphipathic ch aracter of the alpha-helix. The effect of the mutations on virus viabi lity was assayed by transfection of cells with copy RNA transcripts. T he effect on positive-strand RNA replication was examined by introduct ion of the mutations in a subgenomic luciferase replicon and analysis of luciferase accumulation following the transfection of BGM. cells wi th 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 ei ther one of these features caused defects in viral RNA synthesis. in v itro 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 cati onic amphipathic alpha-helix is a major determinant for a function of protein 2B, and possibly its precursor 2BC, in viral RNA synthesis. Th e potential role of the amphipathic alpha-helix in the permeabilizatio n of cellular membranes is discussed.