Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth

Abstract

The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain N o (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain N o from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.

Study of herpes simplex virus maturation during a synchronous wave of assembly

“…The protease responsible is the product of the HSV UL26 gene, and UL26 inactivation blocks capsid maturation (11,29). ICP35c,d cleavage correlates with condensation of the inner shell to generate small-cored B capsids (29,38) and is also required for maturation of the outer shell, since the major capsid protein VP5 fails to display certain epitopes if ICP35 cleavage is blocked (11,21,22). Small-cored B capsids are probably able to package the viral genome, resulting in expulsion of the scaffold core (13,32) and the formation of C capsids, which contain an irregular electron-dense center.…”

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids

“…Although the mutation in A247S abolishes the release of N 0 , this mutant protease retains the ability to autoprocess at the M site and trans cleave ICP35 (30,53). Neither of these mutant viruses grow on Vero cells but require HSV-1 protease-expressing BMS-MG22 cells for their propagation (13,30). As shown in Table 1, transfection with the wt protease gene (Pra) complemented the growth of both mutants, while the HCMV-HSV chimeric protease (HCMVN 0 / HSVNa) was not able to support the growth of these mutant viruses.…”

“…The N-terminal 247-aa sequence of the HSV-1 protease (N 0 ) is enzymatically active but is insufficient to support viral growth (13,30). This suggests that the C-terminal 388-aa sequence (Na) of the protease may perform a function required to support viral growth, perhaps by providing the specific signal required to localize N 0 to the site of capsid assembly (30).…”

“…The N-terminal 247-aa sequence of the HSV-1 protease (N 0 ) is enzymatically active but is insufficient to support viral growth (13,30). This suggests that the C-terminal 388-aa sequence (Na) of the protease may perform a function required to support viral growth, perhaps by providing the specific signal required to localize N 0 to the site of capsid assembly (30). If localization is the only function of Na, then a chimeric protease consisting of the HCMV catalytic domain (HCMVN 0 ) and HSV-1 Na should retain functionality, since the CMV catalytic domain has the ability to cleave HSV substrates (55).…”

“…The mutation in the m100 virus eliminates synthesis of the functional protease and, therefore, ICP35 cannot be cleaved (13). Although the mutation in A247S abolishes the release of N 0 , this mutant protease retains the ability to autoprocess at the M site and trans cleave ICP35 (30,53). Neither of these mutant viruses grow on Vero cells but require HSV-1 protease-expressing BMS-MG22 cells for their propagation (13,30).…”