Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein

Abstract

The coxsackievirus 2B protein is a small hydrophobic protein (99 amino acids) that increases host cell membrane permeability, possibly by forming homo-multimers that build membrane-integral pores. Previously, we defined the functional role of the two hydrophobic regions HR1 and HR2. Here, we investigated the importance of regions outside HR1 and HR2 for multimerization, increasing membrane permeability, subcellular localization, and virus replication through analysis of linker insertion and substitution mutants. From these studies, the following conclusions could be drawn. ( i) The hydrophilic region ( 58 RNHDD 62) between HR1 and HR2 is critical for multimerization and increasing membrane permeability. Substitution analysis of Asn 61 and Asn 62 demonstrated the preference for short polar side chains (Asp, Asn), residues that are often present in turns, over long polar side chains (Glu, Gln). This finding supports the idea that the hydrophilic region is involved in pore formation by facilitating a turn between HR1 and HR2 to reverse chain direction. ( ii) Studies undertaken to define the downstream boundary of HR2 demonstrated that the aromatic residues Trp 80 and Trp 82, but not the positively charged residues Arg 81, Lys 84, and Lys 86 are important for increasing membrane permeability. ( iii) The N terminus is not required for multimerization but does contribute to the membraneactive character of 2B. ( iv) The subcellular localization of 2B does not rely on regions outside HR1 and HR2 and does not require multimerization. ( v) Virus replication requires both the membrane-active character and an additional function of 2B that is not connected to this activity.