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HSV-1 proteinase substrate is derived from the primary translational product of the herpes virus proteinase, contains the putative cleavage site essential for the proteinase maturational release.

Assembly of the herpesvirus capsid includes a maturational proteolytic cleavage that appears to be an essential step in the formation of infectious virions. One target of this cleavage is the procapsid assembly protein precursor which loses a small portion of its carboxyl end during the process. The proteinase responsible for catalyzing this reaction is encoded by a viral gene that is conserved among the herpes group viruses and contains the gene for its substrate, the assembly protein precursor, as its nested, in-frame, 3'-coterminal half 1,2. McGeoch DJ et al., in 1988 identified the complete sequence of this long unique region in the genome of herpes simplex virus type 1 3.

Structural Characteristics
HSV proteinase was studied by using insertion and deletion mutants in transient transfection assays and plasma desorption mass spectrometry to determine its cleavage site in the mature assembly protein. Results suggest that (i) The enzymatic portion of the proteinase is contained within the amino half of the full-length molecule and includes two highly conserved domains, referred to as CDI and CD2. This finding has been confirmed for both herpes simplex virus (HSV) and human cytomegalovirus (HCMV). (ii) Maturational cleavage at the carboxyl end of the assembly protein precursor is between Ala and Ser in the consensus sequence V/L-X-A I S. This sequence is called the matura-tional cleavage site (M site) and is highly conserved in the homologous proteins of other herpes group viruses 4,5.

Mode of Action
The HSV proteinase precursor is cleaved twice-once at its M site, present near its carboxyl end as a consequence of the nested relationship of the proteinase and assembly protein genes and once at a site called the release cleavage site (R site) near the middle of the proteinase precursor. The R site was predicted to be between Ala and Ser in the consensus sequence Y-V/L-K/Q-A I S near the middle of the proteinase and, like the M-site cleavage sequence, is highly conserved among the homologs of other herpes group viruses. Amino acid sequence analyses of bacterially synthesized products of the proteinase have shown that cleavage occurred at the predicted sites and demonstrated the fidelity and utility of the bacterial system. A third cleavage site with the sequence VEA l AT has been identified near the middle of the HCMV 28-kDa assembling homolog, between CDI and CD2.  It has been proposed to inactivate the enzyme and therefore is called the inactivation site (I site) 5. The proteinase activity of herpes simplex virus type 1 (HSV-1) is encoded by the UL26 open reading frame which produces a 635 amino acid (aa) protein with autocatalytic protease activity. Cleavage of this precursor polypeptide occurs between alanine and serine residues at positions 247±248 and 610±611 to generate the capsid proteins VP24 and VP21, and a C-terminal 25 aa fragment. The major substrate of the proteinase is encoded by a collinear gene designated UL26.5, which species the scaffolding protein VP22a. This polypeptide is also cleaved 25 amino acids from its C terminus 6.


Free the proteinase
, this cleavage may be required to free the proteinase, also called assemblin, from its C-terminal end, thereby enabling it to cleave the assembly protein precursor 5.

Virion maturation, the 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. The complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. HSV-1 protease has distinct functional domains and some of these functions can complement in trans 6.

Substrate specificity, Pulse-chase radiolabeling and site-directed mutagenesis indicated that assemblin is metabolically unstable and is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, the enzymatic amino half of the proteinase precursor. Two amino acids were identified as essential for activity: the single absolutely conserved serine and one of the two absolutely conserved histidines. When the highly conserved glutamic acid (Glu22) was substituted, the proteinase was able to cleave at the M and I sites but not at the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., protein conformation) role for this residue in determining substrate specificity 5.


1.     Liu F, Roizman B (1991). The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate. J. Virol., 65:5149-5156.

2.     Welch AR, Woods AS, McNally LM, Cotter RJ, Gibson W (1991). A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site, and cleavage site. PNAS., 88:10792-10796.

3.     McGeoch DJ, Dalrymple MA, Davison AJ, Dolan A, Frame MC, McNab D, Perry PLJ, Scott JE, Taylor P (1988). The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Virol., 69:1531-1574.

4.     Welch AR, McNally LM, Gibson W (1991). Cytomegalovirus assembly protein nested gene family: four 3-coterminal transcripts encode four in-frame, overlapping proteins. J. Virol., 65:4091-4100.

5.     Welch AR, McNally LM, Hall MR, Gibson W (1993). Herpesvirus Proteinase: Site-Directed Mutagenesis Used To Study Maturational, Release, and Inactivation Cleavage Sites of Precursor and To Identify a Possible Catalytic Site Serine and Histidine. Journal of Virology., 67(12):7360-7372.

6.     Liu F,  Roizman B (1993). Characterization of the protease and other products of amino-terminus-proximal cleavage of the herpes simplex virus 1 UL26 protein. Journal of Virology, 67:1300-1309.

7.     Robertson BJ, McCann PJ 3rd, Matusick-Kumar L, Newcomb WW, Brown JC, Colonno RJ, Gao M (1996). Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids. J Virol., 70(7):4317–4328. 

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