Summary auto-generated
Researchers constructed chimeric coxsackievirus B3 (CBV3) genomes containing poliovirus (PV) 2B protein sequences to identify functional domains required for viral RNA replication. The 2B proteins of these enteroviruses share similar biochemical activities—inhibiting protein secretion and modifying membrane permeability—but differ in sequence. Researchers replaced various segments of CBV3 2B with PV 2B counterparts and tested whether the resulting hybrid proteins could support viral replication. A chimeric genome expressing only the amino-terminal one-third of PV 2B fused with the remainder of CBV3 2B produced viable virus with delayed replication kinetics. In contrast, the complete PV 2B protein failed to support replication, as did genomes expressing hybrids with substituted amphipathic α-helices or the second hydrophobic domain. Analysis confirmed that viral polyprotein synthesis and processing occurred normally in all constructs, indicating defects were specific to the 2B protein's function. These findings suggest that the carboxy-terminal two-thirds of 2B contains sequence-specific determinants required for interactions with other viral replication proteins, distinct from the protein's membrane-modifying activities.
Key findings
- Complete poliovirus 2B protein cannot functionally replace coxsackievirus B3 2B in viral RNA replication despite having similar biochemical activities
- Only the amino-terminal one-third of PV 2B can be tolerated when fused to the remainder of CBV3 2B, producing viable virus with delayed growth
- The carboxy-terminal two-thirds of 2B, containing hydrophobic domains, appears essential for sequence-specific interactions with other viral replication proteins
- Proper polyprotein processing occurs normally in all chimeric constructs, indicating the replication defects are specific to 2B protein function rather than processing errors
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Abstract
The 2B proteins of coxsackievirus and poliovirus (PV) share significant structural similarity and exhibit similar biochemical activities, namely inhibition of protein secretion and modification of membrane permeability. 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 functioning in viral RNA replication, a chimeric cDNA approach was used. Chimeric 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 counterparts. In vitro synthesis and processing of the chimeric polyproteins showed no abnormalities. CBV3 genomes carrying the entire PV 2B gene failed to replicate. A chimeric genome that expressed a hybrid 2B protein consisting of the amino-terminal one-third of PV and the remainder of CBV3 yielded viable viruses. In contrast, a 2B protein consisting of the amino-terminal one-third of CBV3 and the remainder of PV failed to drive replication. These data imply that a sequence- specific interaction with another viral protein is required to drive RNA replication and suggest that the proposed sites of contact reside in the carboxy-terminal two-thirds of 2B. Hybrid genomes in which either the amphipathic alpha-helix or the other hydrophobic domain was replaced failed to replicate. The potential contribution of these domains to the structure and functioning of protein 2B are discussed.