A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation

Abstract

The herpesvirus ubiquitin-specific protease (USP) family, whose founding member was discovered as a protease domain embedded in the large tegument protein of herpes simplex virus 1 (HSV-1), is conserved across all members of the Herpesviridae. Whether this conservation is indicative of an essential function of the enzyme in vivo has not yet been established. As reported here, USP activity is conserved in Marek's disease virus (MDV), a tumorigenic alphaherpesvirus. A single amino acid substitution that abolishes the USP activity of the MDV large tegument protein diminishes MDV replication in vivo, and severely limits the oncogenic potential of the virus. Expression of the USP transcripts in MDV-transformed cell lines further substantiates this hypothesis. The herpesvirus USP thus appears to be required not only to maintain a foothold in the immunocompetent host, but also to contribute to malignant outgrowths.chicken ͉ deubiquitinating enzyme ͉ herpes ͉ Marek's disease virus T he ubiquitin-proteasome system controls cytosolic proteolysis, certain aspects of transcription, antigen presentation via major histocompatibility complex (MHC) class I products, and the trafficking of surface-exposed receptors (1-5). As for many other posttranslational modifications, both ubiquitin conjugation and its reversal by ubiquitin-specific proteases (USPs) determine the biological outcome of the reaction. The enzyme families that catalyze ubiquitin conjugation and removal are quite diverse (6-9). Consequently, bioinformatic analysis is not always adequate to identify novel USPs. To target such enzymes biochemically, we developed activity-based probes for USPs and enzymes that act on ubiquitin-like modifiers (10). These probes are equipped with an affinity handle to allow retrieval and identification of the enzymes targeted by the electrophilic warhead installed at the probe's carboxyl terminus.Through the use of one of these probes, HA-ubiquitin vinylmethylester (HA-UbVME), we identified the large tegument protein of herpes simplex virus 1 (HSV-1), viral protein (VP) 1/2, encoded by the unique-long (U L ) 36 gene, as the source of an active USP. Its sequence showed no obvious similarity to known eukaryotic USPs and failed to yield an obvious signature of residues diagnostic of known cysteine protease families. Nonetheless, sequence comparisons across the Herpesviridae show the presence of a few absolutely conserved residues (Cys, Asp, His, Glu), all of which are consistent with involvement in a potential thiol protease active site. We have, meanwhile, confirmed both the mechanism of action of such ubiquitin-based probes (11)(12)(13)(14)(15) and the identity of the viral cysteine protease domain as an authentic USP by crystallographic analysis of the homologous segment of the murine cytomegalovirus (MCMV) M48 protein (16). Notwithstanding the conservation of the identified USP activity in all herpesviruses, we do not know whether this activity makes a contribution to the replicative success and pathogenicity of herpesviruses in vivo...

Autocatalytic Activity of the Ubiquitin-Specific Protease Domain of Herpes Simplex Virus 1 VP1-2

“…These observations notwithstanding, to date there have been few clues as to the role of the USP activity, and while it can have a dramatic effect in certain circumstances (21,27), no rationale for the high conservation of the USP across viruses with diverse pathways of infection and pathogenesis. Furthermore, apart from cleavage of polyubiquitin chains in vitro, there has been little evidence that the USP can deubiquitinate a substrate and influence its degradation.…”

“…In studies of a similar PRV mutant containing a C26A mutation, virus yields in culture were delayed and lower (10-to 20-fold), and a very significant defect was observed in animal models of neuroinvasion from peripheral tissues (27). In the MDV VP1-2 homologue, it was reported that a single substitution in the active site of the enzyme abolished its ubiquitin cleavage activity in vitro, and while the corresponding mutation in the context of a recombinant virus had a modest though detectable effect on replication (4-fold lower yields and up to 50% reduction ion plaque size), it had a profound effect on in vivo pathogenesis, with a 1,000-to 10,000-fold reduction in viremic levels and a drastic reduction in T cell lymphomas in infected chickens (21).…”

“…This USP protein was also shown to cleave lysine 48-linked polyubiquitin chains, an activity completely dependent upon the conserved C65 core catalytic residue. USP activity has now been demonstrated for the VP1-2 homologues of a number of herpesviruses across the alpha, beta, and gamma families including human cytomegalovirus (HCMV) (23,41), mouse cytomegalovirus (MCMV) (36), Epstein-Barr virus (EBV) (36,42), Marek's disease virus (MDV) (21), and Kaposi's sarcoma-associated herpesvirus (KSHV) (16). Analysis of structural features of the USP domain of MCMV indicates that the herpesvirus class of USPs adopts a papain-like fold that is structurally distinct from what is seen for other deubiquitinating enzymes (36).…”

MicroRNA Profile of Marek's Disease Virus-Transformed T-Cell Line MSB-1: Predominance of Virus-Encoded MicroRNAs

“…Samples of 20 g total RNA were resolved in a 15% polyacrylamide-urea gel and blotted onto a GeneScreen Plus membrane (Perkin-Elmer). DNA oligonucleotides with the exact complementary sequence to selected miRNAs were end labeled with [␥- 32 P]ATP by use of T4 polynucleotide kinase (New England Biolabs, Hertfordshire, United Kingdom) to generate high-specific-activity probes. Hybridization, washing, and autoradiography were carried out as previously described (36,53).…”

“…Some of the functions of Meq associated with oncogenic properties, such as its interaction with CtBP, parallel those of other viral oncogenic sequences, such as adenovirus E1A and Epstein-Barr virus (EBV) nuclear antigens EBNA3A and -3C (6), highlighting the convergent evolution of oncogenic pathways in these viruses. Recent studies have also identified the role of other genes, such as the pp38 (23), viral interleukin-8 (vIL-8) (49), ICP4 (15,38), R-LORF4 (33), UL36 (32), and MDV-encoded telomerase RNA (22,63) genes, in pathogenesis.…”