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The method of substrate phage display was used to select a preferred substrate from three monovalent display libraries using the HSV-1 protease. The display libraries consisted of four random amino acids, six random amino acids, and a biased library containing four amino acids from the P side of the HSV-1 maturation site followed by four random amino acids. A series of consensus peptides was synthesized based upon the results from these screens and tested in peptide cleavage assays. An eight amino acid consensus peptide (LVLASSSF) derived from the phage results was cleaved as efficiently as a 20-mer maturation site peptide. The selected amino acid sequences also allowed the design of a four amino acid paranitroanilide substrate for continuous assay of HSV-1 protease. Similar to HCMV protease, these results define P4 to P1 as a minimal substrate recognition domain for the HSV-1 protease and suggest that P4 to P1 is the minimal substrate domain which all herpesvirus proteases recognize.

The herpes simplex virus type 1 UL12 gene product, alkaline nuclease (AN), appears to be involved in viral DNA processing and capsid egress from the nucleus (Shao, L., Rapp, L. M., and Weller, S. K., Virology 196, 146–162, 1993). Although the HSV-1 AN is not absolutely essential for viral replication in tissue culture, conservation of the AN gene in all herpesviruses suggests an important role in the life cycle of herpesviruses. The counterpart of HSV-1 AN for human cytomegalovirus (HCMV) is the UL98 gene product. To examine whether the HCMV AN could substitute for HSV-1 AN, we performed trans -complementation experiments using a HSV-1 amplicon plasmid carrying the HCMV UL98 gene. Our results indicate (i) HCMV AN can complement the growth of the HSV-1 AN deletion mutant UL12lacZ virus in trans; (ii) a new recombinant virus, UL12laZcUL98/99, appears to be generated by the integration of the HCMV UL98 gene into the HSV-1 UL12lacZ viral genome; (iii) in contrast to its parental HSV-1 UL12lacZ virus, capsids formed in UL12lacZUL98/99 -infected Vero cells were able to transport from the nucleus to the cytoplasm and mature into infectious viruses. Our results demonstrate a functional conservation of AN between HSV-1 and HCMV.