Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Laurent, S. Articles by Rasschaert, D. Search for Related Content PubMed PubMed Citation Articles by Laurent, S. Articles by Rasschaert, D.

 Previous Article  |  Next Article 

Journal of Virology, June 2007, p. 6117-6121, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02679-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Sequential Autoprocessing of Marek's Disease Herpesvirus Protease Differs from That of Other Herpesviruses

S. Laurent,^1* C. Blondeau,¹ M. Belghazi,³ S. Remy,¹ E. Esnault,¹ P. Rasschaert,¹ and D. Rasschaert^1,2

Equipe Télomérase et Lymphome Viro-induit, UPR INRA 1282 IASP-213, INRA de Tours, 37380 Nouzilly, France,¹ UFR Sciences et Techniques de l'Université François Rabelais de Tours, 37200 Tours, France,² Service de Spectrométrie de Masse pour la protéomique, Unité PRC, INRA de Tours, 37380 Nouzilly, France³

Received 5 December 2006/ Accepted 14 March 2007

Top ABSTRACT TEXT REFERENCES

 
Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)_P3-X_P2-A_P1/S_P1' (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: M_P3-N_P2-A_P1/S_P1'. Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.

Top ABSTRACT TEXT REFERENCES

 
Marek's disease virus (MDV), the causative agent of a type of T lymphoma in chickens, is a herpesvirus classified as gallid herpesvirus 2 in the Mardivirus genus of the Alphaherpesvirinae subfamily. Marek's disease is one of the most important and widespread infectious diseases of chickens, despite the availability of antitumor vaccines (11).

As with other herpesviruses, MDV virions contain multiple layers, which are assembled in several stages. The spherical precursor capsid, the procapsid, consists of four proteins surrounding an internal scaffold. It forms a major, transient intermediate in the capsid maturation pathway in vivo (14). The scaffold is composed of the products of two overlapping genes (UL26 and UL26-5 in herpes simplex virus type 1) encoding proteins with identical C termini. The N-terminal part of UL26 corresponds to one of the two proteases produced by herpesviruses. During capsid maturation, the UL26-encoded protease precursor undergoes autoprocessing, releasing the N-terminal protease domain by cleavage at the R site. This domain undergoes further autoprocessing and then cleaves the UL26-5-encoded protein at an additional site (M), near the C terminus, releasing a 25-amino-acid peptide that tethers the scaffold to the capsid shell. This step coincides with genome packaging; it is the final step in capsid maturation and is therefore a key stage in capsid assembly (21).

The herpesvirus protease, which belongs to a new class of serine proteases with a specific Ser-His-His catalytic triad (18), is highly conserved (up to 90%) among members of one herpesvirus subfamily (12). A naturally occurring cleavage sequence has been identified for the R and M sites: (V, L, I)-X-A/S, where X is a polar amino acid.

The catalytic triad (His₄₉-Ser₁₁₆-His₁₃₅) and the cleavage sites R (232-L_P3-Q_P2-A_P1/S_P1'-235) and M (636-M_P3-N_P2-A_P1/S_P1'-639) were identified by aligning the predicted amino acid sequence of the polyprotein of MDV with the analogous polyproteins of alphaherpesviruses. Surprisingly, the predicted M site does not match the consensus sequence, differing at M_P3. We characterized the MDV protease cleavage sites by production of the wild-type polyprotein (wtPP) of MDV RB1B and a series of mutants with modified catalytic and cleavage sites by use of a pET-14 expression system (Novagen).

The wtPP gene was amplified by PCR using the primers listed in Table 1 and the PBS library, as previously described (8). It was inserted into the appropriate restriction sites of pET-14 to generate pET-UL26, which was used as a template for PCR mutagenesis and as a subcloning vector for mutated DNA fragments. Mutations were introduced at appropriate locations, employing the overlapping PCR technique (6) with the primers listed in Table 1. Western blot immunodetection of the proteins was carried out with monoclonal antibodies (MAb) against the protease (8B6) and the scaffold protein (FD6) by use of a passive blotting protocol that allows the analysis of two identical symmetrical immunoblots (1). MAb were produced as previously described (15), using His-Bind resin-purified polypeptides produced in the pET-22 system (Novagen) (Table 1) as an antigen.

View this table: [in this window] [in a new window]

TABLE 1. Primers used for pET construction

We assessed the ability of the MDV polyprotein to undergo autoprocessing in Escherichia coli by comparing the MDV wtPP with an S116D mutant harboring mutations in the active site of the enzyme, predicted to result in an absence of catalytic activity (4, 17, 19) (Fig. 1A). As expected, no cleavable product was detected with the S116D mutant; this is consistent with an absence of processing at the R and M sites (Fig. 1B and C). By contrast, with the wtPP, products, most likely cleaved at R, were detected with the antiprotease MAb 8B6 or the anti-scaffold protein MAb FD6.The comparison between the products of cleavage revealed by FD6 and the Sc control (where Sc represents construction of the scaffold protein from the predicted R to the carboxyl end) seems to indicate that the product of scaffolding could correspond to a total cleavage on the level of the predicted sites R and M, considering the observed shift of migration between the wild type and the Sc control (predicted carboxyl fragment of 2,655 Da removed). Autoprocessing of the MDV polyprotein seems to occur in the E. coli expression system, which is consistent with findings for other herpesviruses (Fig. 1B and C) (3, 16).

View larger version (37K): [in this window] [in a new window]

FIG. 1. Comparison of autoprocessing of the wild-type MDV protease precursor and that of precursors with mutations affecting the catalytic and cleavage sites (R and M). (A) Schematic representation of the protease polyprotein, with its R and M cleavage sites indicated. Hatched and gray boxes indicate the protease part of the protein and the matured scaffold protein, respectively. A part of the amino acid sequences of all constructs, with each mutation being framed by a rectangle, is indicated below the scheme. (B) Diagram of cleavage products, with their calculated molecular masses shown. (C) Western blots of proteins corresponding to the constructs expressed in E. coli 3 h after induction (with 1 mM IPTG [isopropyl-ß-D-thiogalactopyranoside]). Two blots, corresponding to the same sodium dodecyl sulfate-polyacrylamide gel (1), were probed with the antiprotease MAb 8B6 (left) and the anti-scaffold protein MAb FD6 (right). The molecular mass of the marker and that of the immunodetected polypeptide are indicated to the right and the left of each blot, respectively. ns, nonspecific band from E. coli; Ctrl, negative control (E. coli). (D) Kinetics of hydrolysis of wtPP. Samples of the wtPP expressed in E. coli were collected at various times after induction with 1 mM IPTG and analyzed by Western blotting. (Left) Analysis of five time points of the kinetic assay. The blot was probed with the antiprotease MAb 8B6. (Right) Analysis of three time points of the kinetic assay. The blot was probed with the anti-scaffold protein FD6.

To confirm the positions of the predicted R and M cleavage sites, we generated two mutants, the A234R and A638R mutants. As with the wtPP, R cleavage products were immunodetected for the A638R mutant (Fig. 1C). Two different scaffold peptides were detected for the wtPP and the A638R mutant, possibly corresponding to scaffold proteins cleaved in M and not cleaved at M, respectively (Fig. 1B), and comparison of the A638R mutant with the scaffold control seems to confirm this. These results are therefore consistent with the predicted site acting as a genuine atypical M site.

The Western blot profile obtained with the A234R mutant was identical to that for the S116D mutant, with one immunoreactive band of 75 kDa (Fig. 1B and C). Surprisingly, no polyprotein cleaved at M (72-kDa band) was detected, whereas such polyproteins are generally detected with other herpesviruses (Fig. 1B) (5, 7, 8, 9, 16, 19).

We therefore carried out liquid chromatography-mass spectrometry experiments to confirm the peptide profiles obtained. Protein bands were excised manually from electrophoresis gels stained with Coomassie blue, subsequently reduced and alkylated, and finally subjected to in-gel proteolysis with trypsin or Lys-C. All digested proteins were sequenced by nano-liquid chromatography-tandem mass spectrometry (Q-TOF-Ultima Global equipped with a nano-electrospray ionization source, coupled with Cap LC nanoHPLC; Waters Micromass). Data were interpreted using ProteinLynx global server software (Waters Micromass). All immunoreactive polypeptides were clearly identifiable, with a coverage of 5 to 73% (Table 2), confirming our identification of the R and M sites. We also confirmed that the 75-kDa product corresponds to the uncleaved protein. Thus, MDV seems to differ from other herpesviruses in that invalidation of the R site prevents cleavage of the M site, indicating that the MDV M site may be cleaved exclusively by the processed short protease domain.

View this table: [in this window] [in a new window]

TABLE 2. Identification of the C-terminal ends of MDV protease polypeptides by mass spectrometry

To confirm this hypothesis, we carried out a kinetic study with the wtPP construct, collecting protein lysate samples at various times after induction (Fig. 1D). The R site was rapidly cleaved, as both the processed short protease and the scaffold protein were detectable 3 min after induction. While the 75-kDa uncleaved polyprotein was immunodetected in all samples, the 72-kDa polyprotein cleaved at M was not detectable until 2 h after induction. Given the strong signal seen at this time in the immunoblot revealed by 8B6, a high concentration of processed short protease seems to be necessary for cleavage of the M site of the polyprotein. However, scaffold protein cleavage in M occurs early (i.e., 3 min postinduction), demonstrating a very rapid cleavage of the M site of this processed substrate.

The lack of M site cleavage when the R site was not cleavable may be due to insufficient recognition of the atypical MDV M site by the full-length protease. We tested this hypothesis by replacing the nonconsensual M_P3 in the MDV M site with the consensual V_P3 of other alphaherpesviruses. First, we confirmed that this modified MDV M site was fully cleaved in the M636V mutant (Fig. 2), demonstrating that the M636V substitution did not prevent M cleavage. We then introduced the M636V mutation into the mutant with the invalidated R site (the A234R mutant) to generate the A234R M636V mutant. Two bands, at 75 kDa and 72 kDa, were detected on Western blots, suggesting that both the full-length precursor and the polyprotein cleaved at the M site were present, as confirmed by mass spectrometry (Table 2; Fig. 2). Thus, the M site was recognized by the full-length protease once the methionine at P3 had been replaced with a valine residue, confirming the importance of the P3 position in the herpesvirus cleavage site, as demonstrated in previous studies for herpes simplex virus type 1 (9, 10) and cytomegalovirus (13, 19).

View larger version (17K): [in this window] [in a new window]

FIG. 2. Mutation of the P3 amino acid of cleavage sites R and M of the MDV protease precursor. (A) Schematic representation of the protease polyprotein, with its R and M cleavage sites indicated. Hatched and gray boxes indicate the protease part of the protein and the matured scaffold protein, respectively. A part of the amino acid sequences of all constructs, with each mutation being framed by a rectangle, is indicated below the scheme. (B) Diagram of cleavage products, with their calculated molecular masses shown. (C) Western blots of proteins corresponding to the constructs expressed in E. coli 3 h after induction (with 1 mM IPTG [isopropyl-ß-D-thiogalactopyranoside]). The blot was probed with the anti-scaffold protein MAb FD6. The molecular mass of the marker and that of the immunodetected polypeptide are indicated to the right and the left of the blot, respectively.

We further investigated the impact of having a methionine residue in the P3 position by introducing M_P3 in the R site (the L232M mutant). Similar Western blot results were obtained for the S116D, A234R, and L232M mutants, with no cleavage at either the R or the M site, demonstrating that a methionine residue at P3 of the R or M site abolishes recognition by the full-length protease (Fig. 2). Furthermore, these data confirmed that R site cleavage was required for M site cleavage, as no 72-kDa product was observed.

Interestingly, these sequential cleavages seem to occur in the order opposite those in other herpesviruses (5, 7, 19). However, in good agreement with observations for the cytomegalovirus protease (20) the full-length protease seemed to have only weak activity, and the lack of cleavage at the M site when the R site was not cleavable may be due to a combination of the weak activity of the polyprotein and poor recognition of the atypical MDV M site by the full-length protease.

As the MDV M site seemed to be cleaved only in trans by the processed short protease, sequential cleavage seems to be necessary, according to the model proposed for Epstein-Barr virus, with three steps and an inactive full-length protease activated by an unknown factor, involving the release of the active short protease (2). Our results also expand the previously described herpesvirus consensus cleavage site from (V, L, I)-X-A/S to (V, L, I, M)-X-A/S.

 
This work was supported by grants from the Ligue Nationale Contre le Cancer-Comité du Cher and the Région Centre, France.

We thank S. Trapp for helpful remarks on the manuscript.

 
* Corresponding author. Mailing address: Equipe Télomérase et Lymphome Viro-induit, UPR INRA 1282 IASP-213, INRA de Tours, 37380 Nouzilly, France. Phone: 33 247427682. Fax: 33 247427774. E-mail: slaurent{at}tours.inra.fr

Published ahead of print on 21 March 2007.

Top ABSTRACT TEXT REFERENCES

 

1
1. Aponte, C., N. M. Mattion, M. K. Estes, A. Charpilienne, and J. Cohen. 1993. Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins. Arch. Virol. 133:85-95.[CrossRef][Medline]
2
2. Buisson, M., J. F. Hernandez, D. Lascoux, G. Schoehn, E. Forest, G. Arlaud, J. M. Seigneurin, R. W. Ruigrok, and W. P. Burmeister. 2002. The crystal structure of the Epstein-Barr virus protease shows rearrangement of the processed C terminus. J. Mol. Biol. 324:89-103.[CrossRef][Medline]
3
3. Deckman, I. C., M. Hagen, and P. J. McCann III. 1992. Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein. J. Virol. 66:7362-7367.[Abstract/Free Full Text]
4
4. Donaghy, G., and R. Jupp. 1995. Characterization of the Epstein-Barr virus proteinase and comparison with the human cytomegalovirus proteinase. J. Virol. 69:1265-1270.[Abstract]
5
5. Godefroy, S., and C. Guenet. 1995. Autoprocessing of HSV-1 protease: effect of deletions on autoproteolysis. FEBS Lett. 357:168-172.[CrossRef][Medline]
6
6. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59.[CrossRef][Medline]
7
7. Jones, T. R., L. Sun, G. A. Bebernitz, V. P. Muzithras, H. J. Kim, S. H. Johnston, and E. Z. Baum. 1994. Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants. J. Virol. 68:3742-3752.[Abstract/Free Full Text]
8
8. Kut, E., and D. Rasschaert. 2004. Assembly of the Marek's disease virus (MDV) capsids using recombinant baculoviruses expressing MDV capsid proteins. J. Gen. Virol. 85:769-774.[Abstract/Free Full Text]
9
9. McCann, P. J., III, D. R. O'Boyle II, and I. C. Deckman. 1994. Investigation of the specificity of the herpes simplex virus type 1 protease by point mutagenesis of the autoproteolysis sites. J. Virol. 68:526-529.[Abstract/Free Full Text]
10
10. O'Boyle, D. R., II, K. A. Pokornowski, P. J. McCann III, and S. P. Weinheimer. 1997. Identification of a novel peptide substrate of HSV-1 protease using substrate phage display. Virology 236:338-347.[CrossRef][Medline]
11
11. Osterrieder, N., J. P. Kamil, D. Schumacher, B. K. Tischer, and S. Trapp. 2006. Marek's disease virus: from miasma to model. Nat. Rev. Microbiol. 4:283-294.[CrossRef][Medline]
12
12. Qiu, X., C. A. Janson, J. S. Culp, S. B. Richardson, C. Debouck, W. W. Smith, and S. S. Abdel-Meguid. 1997. Crystal structure of varicella-zoster virus protease. Proc. Natl. Acad. Sci. USA 94:2874-2879.[Abstract/Free Full Text]
13
13. Sardana, V. V., J. A. Wolfgang, C. A. Veloski, W. J. Long, K. LeGrow, B. Wolanski, E. A. Emini, and R. L. LaFemina. 1994. Peptide substrate cleavage specificity of the human cytomegalovirus protease. J. Biol. Chem. 269:14337-14340.[Abstract/Free Full Text]
14
14. Sheaffer, A. K., W. W. Newcomb, J. C. Brown, M. Gao, S. K. Weller, and D. J. Tenney. 2000. Evidence for controlled incorporation of herpes simplex virus type 1 UL26 protease into capsids. J. Virol. 74:6838-6848.[Abstract/Free Full Text]
15
15. Thouvenin, E., S. Laurent, M. F. Madelaine, D. Rasschaert, J. F. Vautherot, and E. A. Hewat. 1997. Bivalent binding of a neutralising antibody to a calicivirus involves the torsional flexibility of the antibody hinge. J. Mol. Biol. 270:238-246.[CrossRef][Medline]
16
16. Tigue, N. J., and J. Kay. 1998. Active site mutants of human herpesvirus-6 proteinase. FEBS Lett. 441:467-469.[CrossRef][Medline]
17
17. Tigue, N. J., and J. Kay. 1998. Autoprocessing and peptide substrates for human herpesvirus 6 proteinase. J. Biol. Chem. 273:26441-26446.[Abstract/Free Full Text]
18
18. Waxman, L., and P. L. Darke. 2000. The herpesvirus proteases as targets for antiviral chemotherapy. Antivir. Chem. Chemother. 11:1-22.[Medline]
19
19. Welch, A. R., L. M. McNally, M. R. Hall, and W. Gibson. 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. J. Virol. 67:7360-7372.[Abstract/Free Full Text]
20
20. Wittwer, A. J., C. L. Funckes-Shippy, and P. J. Hippenmeyer. 2002. Recombinant full-length human cytomegalovirus protease has lower activity than recombinant processed protease domain in purified enzyme and cell-based assays. Antivir. Res. 55:291-306.[CrossRef][Medline]
21
21. Yu, X., P. Trang, S. Shah, I. Atanasov, Y. H. Kim, Y. Bai, Z. H. Zhou, and F. Liu. 2005. Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy. Proc. Natl. Acad. Sci. USA 102:7103-7108.[Abstract/Free Full Text]

---

Journal of Virology, June 2007, p. 6117-6121, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02679-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Laurent, S. Articles by Rasschaert, D. Search for Related Content PubMed PubMed Citation Articles by Laurent, S. Articles by Rasschaert, D.