GENETIC-ANALYSIS OF A HYDROPHOBIC DOMAIN OF COXSACKIE B3 VIRUS PROTEIN 2B - A MODERATE DEGREE OF HYDROPHOBICITY IS REQUIRED FOR A CIS-ACTING FUNCTION IN VIRAL-RNA SYNTHESIS

Coxsackie B virus protein 2B contains near its C terminus a hydrophobi c domain with an amino acid composition that is characteristic for tra nsmembrane regions. A molecular genetic approach was followed to defin e the role of this domain in virus reproduction and to study the struc tural and hydrophobic requirements of the domain. Nine substitution mu tations were introduced in an infectious cDNA clone of coxsackie B3 vi rus. The effects of the mutations were studied in vivo by transfection of Buffalo green monkey cells with copy RNA transcripts. The results reported here suggest that a critical degree of hydrophobicity of the domain is essential for virus growth. The mutations S77M, C75M 164S, a nd V66S, which caused either a small increase or decrease in mean hydr ophobicity, yielded viable viruses. The double mutations S77M/C75M and I64S/V6-6S, which caused a more pronounced increase or decrease in hy drophobicity, were nonviable. Negatively charged residues (mutations A 71E, I73E, and A71E/I73E) abolished virus growth. The mutations had no effect on the synthesis and processing of the viral polyprotein. Repl ication and complementation were studied by using a subgenomic coxsack ievirus replicon containing the luciferase gene in place of the capsid coding region. Analysis of luciferase accumulation demonstrated that the mutations cause primary defects in viral RNA synthesis that cannot be complemented by wild-type protein 2B provided in trans, The hydrop hobic domain is predicted by computer analysis to form a multimeric tr ansmembrane helix The proposed interaction with the membrane and the i mplications of the mutations on this interaction are discussed.