Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Nov;69(11):7113–7121. doi: 10.1128/jvi.69.11.7113-7121.1995

Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth.

L Matusick-Kumar 1, P J McCann 3rd 1, B J Robertson 1, W W Newcomb 1, J C Brown 1, M Gao 1
PMCID: PMC189631  PMID: 7474131

Abstract

The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain No (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain No from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker T. S., Newcomb W. W., Booy F. P., Brown J. C., Steven A. C. Three-dimensional structures of maturable and abortive capsids of equine herpesvirus 1 from cryoelectron microscopy. J Virol. 1990 Feb;64(2):563–573. doi: 10.1128/jvi.64.2.563-573.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Braun D. K., Roizman B., Pereira L. Characterization of post-translational products of herpes simplex virus gene 35 proteins binding to the surfaces of full capsids but not empty capsids. J Virol. 1984 Jan;49(1):142–153. doi: 10.1128/jvi.49.1.142-153.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casjens S., King J. Virus assembly. Annu Rev Biochem. 1975;44:555–611. doi: 10.1146/annurev.bi.44.070175.003011. [DOI] [PubMed] [Google Scholar]
  5. Deckman I. C., Hagen M., McCann P. J., 3rd Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein. J Virol. 1992 Dec;66(12):7362–7367. doi: 10.1128/jvi.66.12.7362-7367.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Desai P., DeLuca N. A., Glorioso J. C., Person S. Mutations in herpes simplex virus type 1 genes encoding VP5 and VP23 abrogate capsid formation and cleavage of replicated DNA. J Virol. 1993 Mar;67(3):1357–1364. doi: 10.1128/jvi.67.3.1357-1364.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. DiIanni C. L., Drier D. A., Deckman I. C., McCann P. J., 3rd, Liu F., Roizman B., Colonno R. J., Cordingley M. G. Identification of the herpes simplex virus-1 protease cleavage sites by direct sequence analysis of autoproteolytic cleavage products. J Biol Chem. 1993 Jan 25;268(3):2048–2051. [PubMed] [Google Scholar]
  9. Gao M., Knipe D. M. Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein. J Virol. 1989 Dec;63(12):5258–5267. doi: 10.1128/jvi.63.12.5258-5267.1989. [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., Roizman B. Proteins specified by herpes simplex virus. 8. Characterization and composition of multiple capsid forms of subtypes 1 and 2. J Virol. 1972 Nov;10(5):1044–1052. doi: 10.1128/jvi.10.5.1044-1052.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gibson W., Roizman B. Proteins specified by herpes simplex virus. Staining and radiolabeling properties of B capsid and virion proteins in polyacrylamide gels. J Virol. 1974 Jan;13(1):155–165. doi: 10.1128/jvi.13.1.155-165.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heilman C. J., Jr, Zweig M., Stephenson J. R., Hampar B. Isolation of a nucleocapsid polypeptide of herpes simplex virus types 1 and 2 possessing immunologically type-specific and cross-reactive determinants. J Virol. 1979 Jan;29(1):34–42. doi: 10.1128/jvi.29.1.34-42.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holland L. E., Sandri-Goldin R. M., Goldin A. L., Glorioso J. C., Levine M. Transcriptional and genetic analyses of the herpes simplex virus type 1 genome: coordinates 0.29 to 0.45. J Virol. 1984 Mar;49(3):947–959. doi: 10.1128/jvi.49.3.947-959.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacob R. J., Morse L. S., Roizman B. Anatomy of herpes simplex virus DNA. XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA. J Virol. 1979 Feb;29(2):448–457. doi: 10.1128/jvi.29.2.448-457.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knipe D. M., Quinlan M. P., Spang A. E. Characterization of two conformational forms of the major DNA-binding protein encoded by herpes simplex virus 1. J Virol. 1982 Nov;44(2):736–741. doi: 10.1128/jvi.44.2.736-741.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ladin B. F., Blankenship M. L., Ben-Porat T. Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation. J Virol. 1980 Mar;33(3):1151–1164. doi: 10.1128/jvi.33.3.1151-1164.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Liu F. Y., Roizman B. The promoter, transcriptional unit, and coding sequence of herpes simplex virus 1 family 35 proteins are contained within and in frame with the UL26 open reading frame. J Virol. 1991 Jan;65(1):206–212. doi: 10.1128/jvi.65.1.206-212.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Matusick-Kumar L., Hurlburt W., Weinheimer S. P., Newcomb W. W., Brown J. C., Gao M. Phenotype of the herpes simplex virus type 1 protease substrate ICP35 mutant virus. J Virol. 1994 Sep;68(9):5384–5394. doi: 10.1128/jvi.68.9.5384-5394.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. McCann P. J., 3rd, O'Boyle D. R., 2nd, Deckman I. C. Investigation of the specificity of the herpes simplex virus type 1 protease by point mutagenesis of the autoproteolysis sites. J Virol. 1994 Jan;68(1):526–529. doi: 10.1128/jvi.68.1.526-529.1994. [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. Morgenstern J. P., Land H. A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells. Nucleic Acids Res. 1990 Feb 25;18(4):1068–1068. doi: 10.1093/nar/18.4.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Newcomb W. W., Brown J. C. Structure of the herpes simplex virus capsid: effects of extraction with guanidine hydrochloride and partial reconstitution of extracted capsids. J Virol. 1991 Feb;65(2):613–620. doi: 10.1128/jvi.65.2.613-620.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Newcomb W. W., Brown J. C. Use of Ar+ plasma etching to localize structural proteins in the capsid of herpes simplex virus type 1. J Virol. 1989 Nov;63(11):4697–4702. doi: 10.1128/jvi.63.11.4697-4702.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Patel A. H., MacLean J. B. The product of the UL6 gene of herpes simplex virus type 1 is associated with virus capsids. Virology. 1995 Jan 10;206(1):465–478. doi: 10.1016/s0042-6822(95)80062-x. [DOI] [PubMed] [Google Scholar]
  32. Perdue M. L., Cohen J. C., Kemp M. C., Randall C. C., O'Callaghan D. J. Characterization of three species of nucleocapsids of equine herpesvirus type-1 (EHV-1). Virology. 1975 Mar;64(1):187–204. doi: 10.1016/0042-6822(75)90091-4. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Quinlan M. P., Chen L. B., Knipe D. M. The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication. Cell. 1984 Apr;36(4):857–868. doi: 10.1016/0092-8674(84)90035-7. [DOI] [PubMed] [Google Scholar]
  37. Rixon F. J., Cross A. M., Addison C., Preston V. G. The products of herpes simplex virus type 1 gene UL26 which are involved in DNA packaging are strongly associated with empty but not with full capsids. J Gen Virol. 1988 Nov;69(Pt 11):2879–2891. doi: 10.1099/0022-1317-69-11-2879. [DOI] [PubMed] [Google Scholar]
  38. Schrag J. D., Prasad B. V., Rixon F. J., Chiu W. Three-dimensional structure of the HSV1 nucleocapsid. Cell. 1989 Feb 24;56(4):651–660. doi: 10.1016/0092-8674(89)90587-4. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Trus B. L., Newcomb W. W., Booy F. P., Brown J. C., Steven A. C. Distinct monoclonal antibodies separately label the hexons or the pentons of herpes simplex virus capsid. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11508–11512. doi: 10.1073/pnas.89.23.11508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vernon S. K., Ponce de Leon M., Cohen G. H., Eisenberg R. J., Rubin B. A. Morphological components of herpesvirus. III. Localization of herpes simplex virus type 1 nucleocapsid polypeptides by immune electron microscopy. J Gen Virol. 1981 May;54(Pt 1):39–46. doi: 10.1099/0022-1317-54-1-39. [DOI] [PubMed] [Google Scholar]
  43. Weinheimer S. P., McCann P. J., 3rd, O'Boyle D. R., 2nd, Stevens J. T., Boyd B. A., Drier D. A., Yamanaka G. A., DiIanni C. L., Deckman I. C., Cordingley M. G. Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles. J Virol. 1993 Oct;67(10):5813–5822. doi: 10.1128/jvi.67.10.5813-5822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Zhou Z. H., Prasad B. V., Jakana J., Rixon F. J., Chiu W. Protein subunit structures in the herpes simplex virus A-capsid determined from 400 kV spot-scan electron cryomicroscopy. J Mol Biol. 1994 Sep 30;242(4):456–469. doi: 10.1006/jmbi.1994.1594. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES