Inhibition of Human Cytomegalovirus UL80 Protease by Specific Intramolecular Disulfide Bond Formation

Abstract

A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.

Autoprocessing of HSV‐1 protease: effect of deletions on autoproteolysis

“…Prolonged incubation of the translational reaction mixture of pSPTHSVB does not result in any additional processed proteins (Fig. 2B, lane 2), as has been shown for human cytomegalovirus UL80 ORF expressed in E. coli [2]. The third cleavage site in UL80, leading to 2 fragments of 13 and 16 kDa, appears to be specific to cytomegalovirus protease as no form shorter than 247 residues has ever been reported for HSV-I protease.…”

“…Recent reports have demonstrated the involvement of a protease, the role of which is critical for virus maturation. The same maturation mechanism was also reported for cytomegalo [1,2] and pseudorabies viruses [3] and is suspected to be shared by all members of the herpes virus family [1]. The identification and characterisation of a herpes simplex virus gene product required for encapsidation of viral DNA was first described [4] through the isolation of a temperature-sensitive (ts 1201) HSV-1 mutant.…”

“…Translation of the shortest HSV-1 protease corresponding to amino acids 1-247 has not been reported previously. The same type of construct was studied for the HCMV by expression in E. coli by radio-pulse-labelling and by coupled transcription/translation in bacterial $30 extracts [2]. From that study, an autoproteolytic cleavage was clearly demonstrated when experiments were performed by radiolabelling in total E. eoli cells compared to bacterial $30 extracts.…”

“…Several groups have concluded that the proteolytic activity of UL26 ORF is located in the N-terminal part and that two cleavages are necessary to produce the mature protease [9 14]. A third cleavage site in the catalytic region of the protease encoded by human cytomegalovirus UL80 ORF has also been identified [2,15].…”

Selective Nonpeptidic Inhibitors of Herpes Simplex Virus Type 1 and Human Cytomegalovirus Proteases.

“…Several investigations to seek herpesvirus protease inhibitors have recently reported various low molecular weight inhibitors [24][25][26][27][28][29][30][31][32][33] and peptidomimetic inhibitors. 21,34,35) While a low molecular weight inhibitor with in vitro antiviral activity and stability in human plasma has been found, 29) there are likely to be problems involved with the peptidomimetic inhibitors, which display poor cell penetration and are easily subjected to degradation in cell culture.…”