A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation Keith Jarosinski*, Lisa Kattenhorn†, Benedikt Kaufer*, Hidde Ploegh†‡, and Nikolaus Osterrieder*‡ *Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853; and †Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142 Edited by Elliott D. Kieff, Harvard Medical School, Boston, MA, and approved October 29, 2007 (received for review July 5, 2007) The herpesvirus ubiquitin-specific protease (USP) family, whose symptoms and their clockwork timing, as well as the availability founding member was discovered as a protease domain embedded of the natural host, make MDV an ideal model to assess the in the large tegument protein of herpes simplex virus 1 (HSV-1), is importance of the viral USP for alphaherpesvirus pathogenesis conserved across all members of the Herpesviridae. Whether this in vivo. The course of infection includes lytic replication, then conservation is indicative of an essential function of the enzyme in latency, followed by reactivation and the formation of massive T vivo has not yet been established. As reported here, USP activity is cell lymphomas in multiple organs (18–20). We undertook the conserved in Marek’s disease virus (MDV), a tumorigenic alpha- generation of an MDV mutant that lacks the USP activity to herpesvirus. A single amino acid substitution that abolishes the address its role in pathogenicity. USP activity of the MDV large tegument protein diminishes MDV replication in vivo, and severely limits the oncogenic potential of Results the virus. Expression of the USP transcripts in MDV-transformed Characterization of the MDVUSP Active Site Cysteine. In HSV-1, the cell lines further substantiates this hypothesis. The herpesvirus USP UL36-derived USP was discovered as an Ϸ450-aa amino-terminal thus appears to be required not only to maintain a foothold in the fragment of the VP1/2 tegument protein, generated late during viral immunocompetent host, but also to contribute to malignant out- infection (12). So far, only HSV-1 has yielded an enzymatically growths. active, proteolytic fragment that is distinct from the intact tegument protein in virus-infected cells. We cloned and expressed the cor- chicken ͉ deubiquitinating enzyme ͉ herpes ͉ Marek’s disease virus responding region of the MDV UL36 gene (referred to as MD- MICROBIOLOGY VUSP), based on homology and secondary structure predictions (13, he ubiquitin-proteasome system controls cytosolic proteol- 14). This 322-residue fragment, when expressed by in vitro tran- Tysis, certain aspects of transcription, antigen presentation via scription/translation, readily formed adducts with Ub-VME in a major histocompatibility complex (MHC) class I products, and manner sensitive to the inclusion of N-ethylmaleimide (data not the trafficking of surface-exposed receptors (1–5). As for many shown). This is consistent with the involvement of a cysteine residue other posttranslational modifications, both ubiquitin conjuga- in catalysis, similar to its Epstein–Barr virus (EBV), human cyto- tion and its reversal by ubiquitin-specific proteases (USPs) megalovirus (HCMV), MCMV, and HSV-1 counterparts. We determine the biological outcome of the reaction. The enzyme confirmed this by mutagenesis of the putative active site cysteine at position 98 to alanine (MDVUSP/C98A). We further generated families that catalyze ubiquitin conjugation and removal are USP USP/C98A quite diverse (6–9). Consequently, bioinformatic analysis is not recombinant MDV and MDV in Escherichia coli, and always adequate to identify novel USPs. To target such enzymes purified these preparations by means of their carboxyl-terminal His biochemically, we developed activity-based probes for USPs and tags. Although the purified material showed some amino-terminal enzymes that act on ubiquitin-like modifiers (10). These probes heterogeneity (possibly because of the use of alternate translational start sites), covalent adduct formation was unmistakable for the are equipped with an affinity handle to allow retrieval and USP/C98A identification of the enzymes targeted by the electrophilic war- wild-type version, and altogether absent for the MDV head installed at the probe’s carboxyl terminus. mutant (Fig. 1A). We then assayed the enzyme preparations for Through the use of one of these probes, HA-ubiquitin vinyl- hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin (Ub- methylester (HA-UbVME), we identified the large tegument AMC), and only the wild-type enzyme hydrolyzed Ub-AMC (Fig. protein of herpes simplex virus 1 (HSV-1), viral protein (VP) 1/2, 1B). These results demonstrate catalytic activity and establish C98 as the active site cysteine of the MDVUSP/UL36 protein. encoded by the unique-long (UL) 36 gene, as the source of an active USP. Its sequence showed no obvious similarity to known USP eukaryotic USPs and failed to yield an obvious signature of In Vitro Growth of an MDV Active Site Mutant. We used recom- residues diagnostic of known cysteine protease families. None- bination-based mutagenesis of a modified infectious bacterial theless, sequence comparisons across the Herpesviridae show the artificial chromosome (BAC) clone of MDV (pRB-1B), derived from the highly oncogenic RB-1B strain (21), to obtain two presence of a few absolutely conserved residues (Cys, Asp, His, independent isolates of the C98A mutant in the USP domain of Glu), all of which are consistent with involvement in a potential MDV (vC98A-1 and -2). In addition, we prepared revertants of thiol protease active site. We have, meanwhile, confirmed both the mechanism of action of such ubiquitin-based probes (11–15) and the identity of the viral cysteine protease domain as an Author contributions: K.J. and L.K. contributed equally to this work; K.J., L.K., H.P., and N.O. authentic USP by crystallographic analysis of the homologous designed research; K.J., L.K., and B.K. performed research; K.J., L.K., H.P., and N.O. analyzed segment of the murine cytomegalovirus (MCMV) M48 protein data; and K.J., L.K., H.P., and N.O. wrote the paper. (16). Notwithstanding the conservation of the identified USP The authors declare no conflict of interest. activity in all herpesviruses, we do not know whether this activity This article is a PNAS Direct Submission. makes a contribution to the replicative success and pathogenicity Freely available online through the PNAS open access option. of herpesviruses in vivo. ‡To whom correspondence may be addressed. E-mail: [email protected] or no34@ Marek’s disease virus (MDV) is a lymphomagenic alphaher- cornell.edu. pesvirus that affects chickens. It is a major source of concern for This article contains supporting information online at www.pnas.org/cgi/content/full/ the poultry industry and necessitates vaccination of chicken 0706295104/DC1. flocks in ovo or shortly after hatching (17–19). The severity of the © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706295104 PNAS ͉ December 11, 2007 ͉ vol. 104 ͉ no. 50 ͉ 20025–20030 Downloaded by guest on September 25, 2021 USP Fig. 1. Activity of recombinant UL36 . The first 322 residues of MDV UL36 were expressed recombinantly in E. coli and purified by means of a C-terminal His tag. (A) Purified enzyme was incubated in the presence or absence of a molar excess of Ha-UbVME for 15 min at room temperature and subjected to SDS/PAGE. Anti-6-His antibody was used to detect proteins by immunoblot. Unmodified enzyme and covalent adducts are indicated with arrows. A non- specific background protein band is indicated with an asterisk. (B) MDVUSP and Fig. 2. Reduced plaque sizes in vC98A viruses. CKC (A) or CEC (B) cultures MDVUSP/C98A (100 pM) were incubated with a 1,000-fold excess of Ub-AMC (100 were infected with 100 plaques per well of a six-well dish with vRB-1B, nM). Hydrolysis was measured as an increase in fluorescence, indicating vC98A-1, or vC98A-1R. Five days p.i., wells were fixed with 90% cold acetone release of AMC, over time. Filled circles are representative of MDVUSP activity, for 10 min and air dried, and plaques were immunohistochemically stained by and open circles are representative of MDVUSP/C98A activity. using anti-MDV polyclonal antibodies and anti-chicken secondary antibodies labeled with Alexa Fluor 568 (Molecular Probes). Digital images were ob- tained from 75–100 individual plaques by using an epifluorescence Axiovert each of the mutants (vC98A-1R and -2R), in which we restored 25 inverted microscope and an AxioCam HRc digital camera (Zeiss). Plaque the originally mutated sequences to the wild-type parental areas were measured by using the NIH ImageJ software, and shown are the sequence. Analysis of the in vitro growth properties in primary average plaque areas in calibrated units for each respective virus. The average Ϸ chicken embryo cells (CEC) revealed an Ϸ20% reduction in area of plaques produced by the vC98A-1 was significantly smaller ( 50% for CKC and Ϸ25% for CEC) to the wild-type vRB-1B or revertant vC98A-1R (P Ͻ plaque size [Fig. 2B (50% when plated on primary kidney 0.01) by using Student’s t tests. Shown below the histogram are representative epithelial cells; Fig. 2A]. The revertants, as expected, replicated plaques corresponding to the average plaque size of each virus. in a manner indistinguishable from parental vRB-1B upon infection of chicken embryo fibroblast cells (CEC) or chicken kidney cell (CKC) cultures. When endpoint titers from infected 7 days p.i., viremia levels were reduced 1,000- to 10,000-fold for cultures were measured, vC98A-1 grew to titers that were the USP-defective mutants when compared with parental marginally (Ϸ4-fold) lower when compared with the vC98A-1R revertant virus (vC98A-1, 0.6 Ϯ 0.2 ϫ 104; vC98A-1R, 2.2 Ϯ vRB-1B or their respective revertant viruses (Fig.