ASSEMBLY OF THE HERPES-SIMPLEX VIRUS CAPSID - REQUIREMENT FOR THE CARBOXYL-TERMINAL 25 AMINO-ACIDS OF THE PROTEINS ENCODED BY THE UL26 AND UL26.5 GENES

Herpes simplex virus type 1 (HSV-1) intermediate capsids are composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, and the genes that encode these proteins, UL19, UL38, UL26, UL26.5, UL18, UL26, and UL35, respectively. The UL26 gene encodes a protease that cl eaves itself and the product of the UL26.5 gene at a site (M site) 25 amino acids from the C terminus of these two proteins, In addition, th e protease cleaves itself at a second site (R site) between amino acid s 247 and 248. Cleavage of the UL26 protein gives rise to the capsid p roteins VP21 and VP24, and cleavage of the UL26 protein gives rise to the capsid protein VP22a. Previously we described the production of HS V-1 capsids in insect cells by infecting the cells with recombinant ba culoviruses expressing the six capsid genes (D. R. Thomsen, L. L. Roof , and P. L. Homa, J. Virol. 68:2442-2457, 1994). Using this system, we demonstrated that the products of the UL26 and/or UL26.5 genes are re quired as scaffolds for assembly of HSV-1 capsids. To better understan d the functions of the UL26 and UL26.5 proteins in capsid assembly, we constructed baculoviruses that expressed altered UL26 and UL26.5 prot eins, The ability of the altered UL26 and UL26.5 proteins to support H SV-1 capsid assembly was then tested in insect cells. Among the specif ic mutations tested were (i) deletion of the C-terminal 25 amino acids from the proteins coded for by the UL26 and UL26.5 genes; (ii) mutati on of His-61 of the UL26 protein, an amino acid required for protease activity; and (iii) mutation of the R cleavage site of the UL26 protei n, Analysis of the capsids formed with wild-type and mutant proteins s upports the following conclusions: (i) the C-terminal 25 amino acids o f the UL26 and UL26.5 proteins are required for capsid assembly; (ii) the protease activity associated with the UL26 protein is not required for assembly of morphologically normal capsids; and (iii) the uncleav ed forms of the UL26 and UL26.5 proteins are employed in assembly of 1 25-nm-diameter capsids; cleavage of these proteins occurs during or su bsequent to capsid assembly. Finally, we carried out in vitro experime nts in which the major capsid protein VP5 was mixed with wild-type or truncated UL26.5 protein and then precipitated with a VP5-Specific mon oclonal antibody. Analysis of the precipitate by sodium dodecyl sulfat e-polyacrylamide gel electrophoresis showed that the two proteins copr ecipitated only when the UL26.5 gene product contained the C-terminal 25 amino acids. The overall results of these experiments support a mod el for capsid assembly in which the uncleaved forms of the UL26 and UL 26.5 proteins are used to assemble the HSV-1 B capsid. The C-terminal 25 amino acids of the UL26 and UL26.5 proteins are suggested to be dir ectly involved in interaction of the scaffold with VP5 in the capsid s hell.