Proline and tyrosine residues in scaffold proteins of herpes simplex virus 1 critical to the interaction with portal protein and its incorporation into capsids.

Previous results showed that amino acids 449 to 457 of pU(L)26, a component of the scaffold of herpes simplex virus 1 capsids, were critical for interaction with the portal protein encoded by U(L)6 and for incorporation of the portal into capsids. To identify residues in this scaffold domain critical for the interaction with pU(L)6, the two proteins were coexpressed in the absence of other viral proteins and subjected to immunoprecipitation with scaffold-specific antibody. Coimmunoprecipitation of pU(L)6 was precluded by pU(L)26 mutations Y451A, P452A, and E454A but not by P449A, R456A, or Y450A. In infected cells, Y451A and P452A diminished solubilization of pU(L)6, reduced incorporation of the portal into the capsid, and precluded viral replication and DNA packaging. In contrast, E454A did not affect these parameters despite the fact that E454 is invariant in a number of different alphaherpesvirus scaffold proteins. These data suggest that the interaction between the scaffold E454A mutant and portal protein is rescued by other viral functions. Finally, we show that amino acids 448 to 459 of pU(L)26 are sufficient to interact with pU(L)6 in a glutathione S-transferase pulldown assay in the absence of other viral proteins and that this interaction is inhibited with excess peptide containing pU(L)26 amino acids 443 to 462. Together, these observations suggest that a direct interaction between this scaffold domain and portal protein mediates incorporation of the portal into the capsid.