Summary auto-generated
This study characterizes the herpes simplex virus type 1 (HSV-1) proteinase and its substrate using newly developed monoclonal antibodies against VP24, a capsid protein product of the UL26 gene. The researchers generated ten anti-VP24 monoclonal antibodies and used them to detect two polypeptide forms of VP24 in infected cells and capsids, related by disulfide bonding. Western blot analysis of different capsid types revealed that VP24 dimers appeared specifically in B capsids under non-reducing conditions, suggesting disulfide-linked dimers may represent the active form of the proteinase. Subcellular fractionation experiments demonstrated that full-length and C-terminal cleaved UL26 products were absent from cell nuclei at early infection times, while only cleaved forms of both UL26 and UL26.5 gene products accumulated in the nucleus. The findings suggest that the HSV-1 proteinase may be activated through disulfide bond-mediated dimerization within assembling capsids in the nucleus, providing a potential temporal control mechanism for proteolytic activity during viral particle assembly.
Key findings
- VP24 exists as two polypeptide forms related by disulfide bonding
- VP24 dimers were detected in B capsids (sites of proteinase action) only under non-reducing conditions, suggesting disulfide-linked dimers represent the active proteinase form
- Only cleaved forms of UL26 and UL26.5 gene products accumulated in cell nuclei, not full-length precursors
- The monoclonal antibodies recognize epitopes in the N-terminal 75 amino acids of VP24
- Disulfide bond-mediated dimerization may provide temporal control of protease activity during capsid assembly
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Abstract
The herpes simplex virus type 1 (HSV-1) capsid protein VP24 (encoded by UL26) was expressed as a GST-fusion protein and used to prepare a group of monoclonal antibodies. These were used to characterize the protein in capsids and virus infected cells and demonstrated that it exists as two polypeptide species. The nature of the relationship between these two species was investigated and found to be associated with disulphide bonding. Under non-reducing conditions a species corresponding to dimers of VP24 was identified in preparations of B capsids, the site of action of the proteinase. Biochemical subcellular fractionation studies suggested that only cleaved forms of UL26 and UL26.5 gene products could be detected in the nucleus of the infected cell at early times post-infection.