RELEASE OF THE CATALYTIC DOMAIN N-O FROM THE HERPES-SIMPLEX VIRUS TYPE-1 PROTEASE IS REQUIRED FOR VIRAL GROWTH

The herpes simplex virus type 1 (HSV-1) protease and its substrate, IC P35, are involved in the assembly of viral capsids and required for ef ficient viral growth. The full-length protease (Pra) consists of 635 a mino acid (aa) residues and is autoproteolytically processed at the re lease (R) site and the maturation (M) site, releasing the catalytic do main N-o (VP24), Nb (VP21), and a 25-aa peptide. To understand the bio logical importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were perfor med to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mut ant m100. Our results indicate that (i) expression of full-length prot ease is not required for viral replication, since a 514-aa protease mo lecule lacking the M site could support viral growth; and that (ii) el imination of the R site by changing the residue Ala-247 to Ser abolish ed viral replication. To better understand the functions that are medi ated by proteolytic processing at the R site of the protease, we engin eered an HSV-I recombinant virus containing a mutation at this site. A nalysis of the mutant A247S virus demonstrated that (i) the mutant pro tease retained the ability to cleave at the M site and to trans proces s ICP35 but failed to support viral growth on Vero cells, demonstratin g that release of the catalytic domain N-o from Pra is required for vi ral replication; and that (ii) only empty capsid structures were obser ved by electron microscopy in thin sections of A247S-infected Vero cel ls, indicating that viral DNA was not encapsidated. Our results demons trate that processing of ICP35 is not sufficient to support viral repl ication and provide genetic evidence that the HSV-1 protease has nucle ar functions other than enzymatic activity.