CHIMERIC COXSACKIE-B3-VIRUS GENOMES THAT EXPRESS HYBRID COXSACKIEVIRUS-POLIOVIRUS-2B PROTEINS - FUNCTIONAL DISSECTION OF STRUCTURAL DOMAINSINVOLVED IN RNA REPLICATION

The 2B proteins of coxsackievirus and poliovirus (PV) share significan t structural similarity and exhibit similar biochemical activities, na mely inhibition of protein secretion and modification of membrane perm eability, Both proteins contain two hydrophobic domains in the carboxy -terminal two-thirds of their sequence, of which one has the potential to form a cationic amphipathic alpha-helix. To gain more insight into the structural requirements of enterovirus protein 2B for its functio ning in viral RNA replication, a chimeric cDNA approach was used. Chim eric coxsackie B3 virus (CBV3) genomes were constructed that expressed either the entire PV 2B protein or hybrid proteins in which specific segments of CBV3 2B were substituted by their corresponding PV counter parts, In vitro synthesis and processing of the chimeric polyproteins showed no abnormalities, CBV3 genomes carrying the entire PV 2B gene f ailed to replicate. Achimeric genome that expressed a hybrid 2B protei n consisting of the amino-terminal one-third of PV and the remainder o f CBV3 yielded viable viruses, in contrast, a 2B protein consisting of the amino-terminal one-third of CBV3 and the remainder of PV failed t o drive replication. These data imply that a sequence-specific interac tion with another viral protein is required to drive RNA replication a nd suggest that the proposed sites of contact reside in the carboxy-te rminal two-thirds of 2B, Hybrid genomes in which either the amphipathi c alpha-helix or the other hydrophobic domain was replaced failed to r eplicate. The potential contribution of these domains to the structure and functioning of protein 2B are discussed.