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 pUL26, a component of the scaffold of herpes simplex virus 1 capsids, were critical for interaction with the portal protein encoded by UL6 and for incorporation of the portal into capsids. To identify residues in this scaffold domain critical for the interaction with pUL6, the two proteins were coexpressed in the absence of other viral proteins and subjected to immunoprecipitation with scaffold-specific antibody. Coimmunoprecipitation of pUL6 was precluded by pUL26 mutations Y451A, P452A, and E454A but not by P449A, R456A, or Y450A. In infected cells, Y451A and P452A diminished solubilization of pUL6, 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 pUL26 are sufficient to interact with pUL6 in a glutathione S-transferase pulldown assay in the absence of other viral proteins and that this interaction is inhibited with excess peptide containing pUL26 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. All DNA-containing herpesvirus capsids contain an oute