A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.

Abstract

A herpesvirus proteinase activity has been identified and partially characterized by using the cloned enzyme and substrate genes in transient transfection assays. Evidence is presented that the proteinase gene of cytomegalovirus strain Colburn encodes a 590-amino acid protein whose N-terminal 249 residues contain the proteolytic activity and two domains that are highly conserved in the homologous protein ofother herpesviruses. Insertion ofa short amino acid sequence between these domains abolished proteinase activity, suggesting that this region constitutes part or all of the enzyme active site. Plasma desorption mass spectrometry was used to identify the C terminus of the mature assembly protein as alanine, enabling the recognition of a consensus proteinase cleavage sequence of V/L-X-A 4 S/V, near the C-terminal end of all herpesvirus assembly protein homologs. Interestingly, the proteinase and its substrate, the assembly protein precursor, are encoded by opposite halves of the same open reading frame.

In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

“…However, the minimal length of a peptide substrate for the HSV-1 protease is large, requiring 13 amino acids [20] and the minimal length of a peptide substrate for HCMV has been established for only the maturation cleavage site [21]. In addition, several reports have suggested that the HCMV protease belongs to either the serine or cysteine class of proteases [5,8,10,111 and no direct biochemical evidence has been reported. Recently, the HSV-1 protease has been classified as a serine protease by identifying the residue at the active site as Ser129 [23].…”

“…As a consequence, the assembly protein is proposed to play a role in virus maturation analogous to that of the scaffolding protein of bacteriophages [4]. During infection, the HCMV preassembly protein undergoes proteolytic processing to form the assembly protein, a lower molecular-mass species that lacks 64 amino acids at the carboxy terminus [5]. Mutants of HSV-1, defective in processing the HSV-1 assembly protein, form aberrant empty capsids and fail to package DNA, indicating that proteolytic processing of the virus assembly protein is critical for herpes virus particle maturation [6, 71. The protease responsible for processing the preassembly protein during HCMV infection consists of 708 amino acids encoded by the UL80 open reading frame, which is 3' coterminal with the gene encoding the preassembly protein [5,8,91.…”

“…The HCMV protease also undergoes autoproteolytic processing at a site (release) closer to its amino-terminus [ 5 , 81 between Ala256 and Ser257. Studies have demonstrated that the catalytic domain of the protease is contained in the first 256 amino acids of the protease [5,8,10, 111, and is released by the release cleavage. A third autoprocessing site has been identified in the catalytic domain of HCMV protease, at Ala143-Ala144 and is proposed to inactivate the enzyme [8,11, 121.…”

“…herpes simplex virus type 1; SCMV, simian cytomegalovirus. encoded by the UL80 open reading frame, which is 3' coterminal with the gene encoding the preassembly protein [5,8, 91. Consequently, the 373 amino acids of the preassembly protein are identical to the carboxy-terminal 373 amino acids of the protease [9] and the protease is capable of autoproteolytic processing its own carboxy terminus at a site identical to that of its substrate (maturational site) between Ala643 and Ser644.…”

“…During infection, the HCMV preassembly protein undergoes proteolytic processing to form the assembly protein, a lower molecular-mass species that lacks 64 amino acids at the carboxy terminus [5]. Mutants of HSV-1, defective in processing the HSV-1 assembly protein, form aberrant empty capsids and fail to package DNA, indicating that proteolytic processing of the virus assembly protein is critical for herpes virus particle maturation [6, 71. The protease responsible for processing the preassembly protein during HCMV infection consists of 708 amino acids encoded by the UL80 open reading frame, which is 3' coterminal with the gene encoding the preassembly protein [5,8,91. Consequently, the 373 amino acids of the preassembly protein are identical to the carboxy-terminal 373 amino acids of the protease [9] and the protease is capable of autoproteolytic processing its own carboxy terminus at a site identical to that of its substrate (maturational site) between Ala643 and Ser644.…”