SEPARATE FUNCTIONAL DOMAINS OF THE HERPES-SIMPLEX VIRUS TYPE-1 PROTEASE - EVIDENCE FOR CLEAVAGE INSIDE CAPSIDS

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related pro teins are involved in the assembly of vital capsids and virion maturat ion. Pra is a serine protease, and the active-site residue has been ma pped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by th e UL26 gene, is autoproteolytically processed at two sites, the releas e (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itsel f at both sites, it releases Nb, the catalytic domain (N-0), and the C -terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the prod uct of the UL26.5 gene. As it is translated from a Met codon within th e UL26 gene, ICP35 c,d are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully professed Pra ( N-0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A2 47S virus, we have recently shown that this mutant protease retains en zymatic activity but fails to support viral growth, suggesting that th e release of N-0 is required for viral replication. Here we report tha t another mutant protease, with an amino acid substitution (Ser to Cys ) at the active site, can complement the A2475 mutant but not a protea se deletion mutant. Cell lines expressing the active-site mutant prote ase were isolated and shown tb complement the A247S mutant at the leve ls of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic c omplementation is that following sedimentation of infected-cell lysate s on sucrose gradients, both N-terminally unprofessed and processed pr oteases were isolated from the fractions where normal B capsids sedime nt, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional do mains and some of these functions can complement in trans.