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Cross-functional analysis of the Microviridae internal scaffolding protein
Abstract:
The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins. These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle. The genes encoding three Microviridae (phiX174, G4 and alpha3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. The phiX174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21 degreesC. Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction. Two G4 mutants, which can productively utilize the phiX174 B protein at elevated temperatures, were isolated. Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region.
Insights
Microviridae scaffolding proteins (B proteins) can assemble foreign viral proteins into infectious virions, suggesting inherent flexibility. However, phiX174 B protein function is temperature-sensitive with G4 capsid assembly.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Scaffolding proteins mediate viral structural protein assembly into infectious virions.
- These proteins are transiently involved in morphogenesis and absent in mature particles.
Purpose of the Study:
- To investigate the cross-complementation capabilities of Microviridae internal scaffolding proteins (B proteins).
- To explore the functional flexibility and interaction dynamics of these proteins.
Main Methods:
- Cloning and in vivo expression of B protein genes from phiX174, G4, and alpha3.
- Assaying complementation of null mutations across different Microviridae species.
- Isolation and characterization of temperature-sensitive G4 mutants.
Main Results:
- Significant cross-complementation observed between divergent Microviridae B proteins, indicating functional flexibility.
- phiX174 B protein failed to complement G4 capsid assembly above 21°C.
- G4 mutants with coat protein substitutions showed improved utilization of phiX174 B protein at elevated temperatures.
Conclusions:
- Microviridae internal scaffolding proteins exhibit inherent flexibility in directing viral assembly.
- Specific coat-scaffolding interactions, particularly in conserved regions, are crucial for efficient assembly, especially under restrictive conditions.