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. 1996 Jul;70(7):4819–4824. doi: 10.1128/jvi.70.7.4819-4824.1996

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics.

R L LaFemina 1, K Bakshi 1, W J Long 1, B Pramanik 1, C A Veloski 1, B S Wolanski 1, A I Marcy 1, D J Hazuda 1
PMCID: PMC190425  PMID: 8676515

Abstract

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.

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Selected References

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  1. Albrecht J. C., Nicholas J., Biller D., Cameron K. R., Biesinger B., Newman C., Wittmann S., Craxton M. A., Coleman H., Fleckenstein B. Primary structure of the herpesvirus saimiri genome. J Virol. 1992 Aug;66(8):5047–5058. doi: 10.1128/jvi.66.8.5047-5058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  3. Baum E. Z., Bebernitz G. A., Hulmes J. D., Muzithras V. P., Jones T. R., Gluzman Y. Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites. J Virol. 1993 Jan;67(1):497–506. doi: 10.1128/jvi.67.1.497-506.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burck P. J., Berg D. H., Luk T. P., Sassmannshausen L. M., Wakulchik M., Smith D. P., Hsiung H. M., Becker G. W., Gibson W., Villarreal E. C. Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites. J Virol. 1994 May;68(5):2937–2946. doi: 10.1128/jvi.68.5.2937-2946.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  6. Cox G. A., Wakulchik M., Sassmannshausen L. M., Gibson W., Villarreal E. C. Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad. J Virol. 1995 Jul;69(7):4524–4528. doi: 10.1128/jvi.69.7.4524-4528.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davison A. J., Scott J. E. The complete DNA sequence of varicella-zoster virus. J Gen Virol. 1986 Sep;67(Pt 9):1759–1816. doi: 10.1099/0022-1317-67-9-1759. [DOI] [PubMed] [Google Scholar]
  8. Desai P., Watkins S. C., Person S. The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame. J Virol. 1994 Sep;68(9):5365–5374. doi: 10.1128/jvi.68.9.5365-5374.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donaghy G., Jupp R. Characterization of the Epstein-Barr virus proteinase and comparison with the human cytomegalovirus proteinase. J Virol. 1995 Feb;69(2):1265–1270. doi: 10.1128/jvi.69.2.1265-1270.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gao M., Matusick-Kumar L., Hurlburt W., DiTusa S. F., Newcomb W. W., Brown J. C., McCann P. J., 3rd, Deckman I., Colonno R. J. The protease of herpes simplex virus type 1 is essential for functional capsid formation and viral growth. J Virol. 1994 Jun;68(6):3702–3712. doi: 10.1128/jvi.68.6.3702-3712.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson W., Marcy A. I., Comolli J. C., Lee J. Identification of precursor to cytomegalovirus capsid assembly protein and evidence that processing results in loss of its carboxy-terminal end. J Virol. 1990 Mar;64(3):1241–1249. doi: 10.1128/jvi.64.3.1241-1249.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gompels U. A., Nicholas J., Lawrence G., Jones M., Thomson B. J., Martin M. E., Efstathiou S., Craxton M., Macaulay H. A. The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution. Virology. 1995 May 10;209(1):29–51. doi: 10.1006/viro.1995.1228. [DOI] [PubMed] [Google Scholar]
  13. Griffin A. M. The complete sequence of the capsid p40 gene from infectious laryngotracheitis virus. Nucleic Acids Res. 1990 Jun 25;18(12):3664–3664. doi: 10.1093/nar/18.12.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holwerda B. C., Wittwer A. J., Duffin K. L., Smith C., Toth M. V., Carr L. S., Wiegand R. C., Bryant M. L. Activity of two-chain recombinant human cytomegalovirus protease. J Biol Chem. 1994 Oct 14;269(41):25911–25915. [PubMed] [Google Scholar]
  15. Irmiere A., Gibson W. Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology. 1983 Oct 15;130(1):118–133. doi: 10.1016/0042-6822(83)90122-8. [DOI] [PubMed] [Google Scholar]
  16. Irmiere A., Gibson W. Isolation of human cytomegalovirus intranuclear capsids, characterization of their protein constituents, and demonstration that the B-capsid assembly protein is also abundant in noninfectious enveloped particles. J Virol. 1985 Oct;56(1):277–283. doi: 10.1128/jvi.56.1.277-283.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones T. R., Sun L., Bebernitz G. A., Muzithras V. P., Kim H. J., Johnston S. H., Baum E. Z. Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants. J Virol. 1994 Jun;68(6):3742–3752. doi: 10.1128/jvi.68.6.3742-3752.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kennard J., Rixon F. J., McDougall I. M., Tatman J. D., Preston V. G. The 25 amino acid residues at the carboxy terminus of the herpes simplex virus type 1 UL26.5 protein are required for the formation of the capsid shell around the scaffold. J Gen Virol. 1995 Jul;76(Pt 7):1611–1621. doi: 10.1099/0022-1317-76-7-1611. [DOI] [PubMed] [Google Scholar]
  19. Lee J. Y., Irmiere A., Gibson W. Primate cytomegalovirus assembly: evidence that DNA packaging occurs subsequent to B capsid assembly. Virology. 1988 Nov;167(1):87–96. doi: 10.1016/0042-6822(88)90057-8. [DOI] [PubMed] [Google Scholar]
  20. Liu F. Y., Roizman B. The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate. J Virol. 1991 Oct;65(10):5149–5156. doi: 10.1128/jvi.65.10.5149-5156.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liu F., Roizman B. Characterization of the protease and other products of amino-terminus-proximal cleavage of the herpes simplex virus 1 UL26 protein. J Virol. 1993 Mar;67(3):1300–1309. doi: 10.1128/jvi.67.3.1300-1309.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liu F., Roizman B. Differentiation of multiple domains in the herpes simplex virus 1 protease encoded by the UL26 gene. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2076–2080. doi: 10.1073/pnas.89.6.2076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Loutsch J. M., Galvin N. J., Bryant M. L., Holwerda B. C. Cloning and sequence analysis of murine cytomegalovirus protease and capsid assembly protein genes. Biochem Biophys Res Commun. 1994 Aug 30;203(1):472–478. doi: 10.1006/bbrc.1994.2206. [DOI] [PubMed] [Google Scholar]
  24. Matusick-Kumar L., Newcomb W. W., Brown J. C., McCann P. J., 3rd, Hurlburt W., Weinheimer S. P., Gao M. The C-terminal 25 amino acids of the protease and its substrate ICP35 of herpes simplex virus type 1 are involved in the formation of sealed capsids. J Virol. 1995 Jul;69(7):4347–4356. doi: 10.1128/jvi.69.7.4347-4356.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McGeoch D. J., Dalrymple M. A., Davison A. J., Dolan A., Frame M. C., McNab D., Perry L. J., Scott J. E., Taylor P. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988 Jul;69(Pt 7):1531–1574. doi: 10.1099/0022-1317-69-7-1531. [DOI] [PubMed] [Google Scholar]
  26. Nicholson P., Addison C., Cross A. M., Kennard J., Preston V. G., Rixon F. J. Localization of the herpes simplex virus type 1 major capsid protein VP5 to the cell nucleus requires the abundant scaffolding protein VP22a. J Gen Virol. 1994 May;75(Pt 5):1091–1099. doi: 10.1099/0022-1317-75-5-1091. [DOI] [PubMed] [Google Scholar]
  27. O'Boyle D. R., 2nd, Wager-Smith K., Stevens J. T., 3rd, Weinheimer S. P. The effect of internal autocleavage on kinetic properties of the human cytomegalovirus protease catalytic domain. J Biol Chem. 1995 Mar 3;270(9):4753–4758. doi: 10.1074/jbc.270.9.4753. [DOI] [PubMed] [Google Scholar]
  28. Person S., Laquerre S., Desai P., Hempel J. Herpes simplex virus type 1 capsid protein, VP21, originates within the UL26 open reading frame. J Gen Virol. 1993 Oct;74(Pt 10):2269–2273. doi: 10.1099/0022-1317-74-10-2269. [DOI] [PubMed] [Google Scholar]
  29. Preston V. G., Coates J. A., Rixon F. J. Identification and characterization of a herpes simplex virus gene product required for encapsidation of virus DNA. J Virol. 1983 Mar;45(3):1056–1064. doi: 10.1128/jvi.45.3.1056-1064.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Preston V. G., Rixon F. J., McDougall I. M., McGregor M., al Kobaisi M. F. Processing of the herpes simplex virus assembly protein ICP35 near its carboxy terminal end requires the product of the whole of the UL26 reading frame. Virology. 1992 Jan;186(1):87–98. doi: 10.1016/0042-6822(92)90063-u. [DOI] [PubMed] [Google Scholar]
  31. Preston V. G., al-Kobaisi M. F., McDougall I. M., Rixon F. J. The herpes simplex virus gene UL26 proteinase in the presence of the UL26.5 gene product promotes the formation of scaffold-like structures. J Gen Virol. 1994 Sep;75(Pt 9):2355–2366. doi: 10.1099/0022-1317-75-9-2355. [DOI] [PubMed] [Google Scholar]
  32. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  33. Sardana V. V., Wolfgang J. A., Veloski C. A., Long W. J., LeGrow K., Wolanski B., Emini E. A., LaFemina R. L. Peptide substrate cleavage specificity of the human cytomegalovirus protease. J Biol Chem. 1994 May 20;269(20):14337–14340. [PubMed] [Google Scholar]
  34. Steffy K. R., Schoen S., Chen C. M. Nucleotide sequence of the herpes simplex virus type 2 gene encoding the protease and capsid protein ICP35. J Gen Virol. 1995 Apr;76(Pt 4):1069–1072. doi: 10.1099/0022-1317-76-4-1069. [DOI] [PubMed] [Google Scholar]
  35. Stevens J. T., Mapelli C., Tsao J., Hail M., O'Boyle D., 2nd, Weinheimer S. P., Diianni C. L. In vitro proteolytic activity and active-site identification of the human cytomegalovirus protease. Eur J Biochem. 1994 Dec 1;226(2):361–367. doi: 10.1111/j.1432-1033.1994.tb20060.x. [DOI] [PubMed] [Google Scholar]
  36. Tatman J. D., Preston V. G., Nicholson P., Elliott R. M., Rixon F. J. Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses. J Gen Virol. 1994 May;75(Pt 5):1101–1113. doi: 10.1099/0022-1317-75-5-1101. [DOI] [PubMed] [Google Scholar]
  37. Telford E. A., Watson M. S., McBride K., Davison A. J. The DNA sequence of equine herpesvirus-1. Virology. 1992 Jul;189(1):304–316. doi: 10.1016/0042-6822(92)90706-u. [DOI] [PubMed] [Google Scholar]
  38. Thomsen D. R., Newcomb W. W., Brown J. C., Homa F. L. Assembly of the herpes simplex virus capsid: requirement for the carboxyl-terminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes. J Virol. 1995 Jun;69(6):3690–3703. doi: 10.1128/jvi.69.6.3690-3703.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Thomsen D. R., Roof L. L., Homa F. L. Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins. J Virol. 1994 Apr;68(4):2442–2457. doi: 10.1128/jvi.68.4.2442-2457.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Welch A. R., McNally L. M., Gibson W. Cytomegalovirus assembly protein nested gene family: four 3'-coterminal transcripts encode four in-frame, overlapping proteins. J Virol. 1991 Aug;65(8):4091–4100. doi: 10.1128/jvi.65.8.4091-4100.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Welch A. R., McNally L. M., Hall M. R., Gibson W. 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. J Virol. 1993 Dec;67(12):7360–7372. doi: 10.1128/jvi.67.12.7360-7372.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Welch A. R., Woods A. S., McNally L. M., Cotter R. J., Gibson W. A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10792–10796. doi: 10.1073/pnas.88.23.10792. [DOI] [PMC free article] [PubMed] [Google Scholar]

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