Related Experiment Videos
Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids
B J Robertson1, P J McCann, L Matusick-Kumar
1Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492-7660, USA.
Abstract:
The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd 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 processed Pra (N0 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 A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels 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 complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.
Insights
Herpes simplex virus type 1 protease (Pra) functional domains can complement each other in trans. Active-site and release-site mutants of Pra demonstrate intragenic complementation, restoring viral replication and capsid assembly.
Area of Science:
- Virology
- Molecular Biology
- Enzymology
Background:
- Herpes simplex virus type 1 (HSV-1) protease (Pra) is crucial for viral capsid assembly and maturation.
- Pra is a serine protease encoded by the UL26 gene, undergoing autoproteolytic processing at release (R) and maturation (M) sites.
- ICP35, a substrate of Pra, is encoded by the UL26.5 gene and shares sequence identity with the C-terminal portion of Pra.
Purpose of the Study:
- To investigate the functional complementation between different HSV-1 protease mutants.
- To determine the role of the N0 domain release in viral replication.
- To elucidate the mechanism and location of Pra proteolytic processing.
Main Methods:
- Construction and analysis of HSV-1 protease mutants, including an R-site mutant (A247S) and an active-site mutant (Ser to Cys).
- Complementation assays using cell lines expressing mutant proteases.
- Analysis of viral capsid assembly, DNA packaging, and viral growth.
- Sucrose gradient sedimentation of infected-cell lysates to determine protease localization and processing.
Main Results:
- An active-site mutant protease complemented the A247S R-site mutant, restoring wild-type Pra function, including viral growth and capsid assembly.
- Complementation was not observed with a protease deletion mutant, indicating the requirement for specific protease domains.
- Proteolytic processing of HSV-1 protease was found to occur within viral capsids, as evidenced by sedimentation analysis.
Conclusions:
- HSV-1 protease possesses distinct functional domains that can complement each other in trans.
- The release of the N0 domain is essential for viral replication.
- Intragenic complementation between R-site and active-site mutants can reconstitute essential Pra functions, with processing occurring intracellularly within capsids.