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.

Fig. 1.

Activity of recombinant UL36^USP. The first 322 residues of MDV U_L36 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 nonspecific background protein band is indicated with an asterisk. (B) MDV^USP and MDV^USP/C98A (100 pM) were incubated with a 1,000-fold excess of Ub-AMC (100 nM). Hydrolysis was measured as an increase in fluorescence, indicating release of AMC, over time. Filled circles are representative of MDV^USP activity, and open circles are representative of MDV^USP/C98A activity.

Fig. 2.

Reduced plaque sizes in vC98A viruses. CKC (A) or CEC (B) cultures were infected with 100 plaques per well of a six-well dish with vRB-1B, vC98A-1, or vC98A-1R. Five days p.i., wells were fixed with 90% cold acetone for 10 min and air dried, and plaques were immunohistochemically stained by using anti-MDV polyclonal antibodies and anti-chicken secondary antibodies labeled with Alexa Fluor 568 (Molecular Probes). Digital images were obtained from 75–100 individual plaques by using an epifluorescence Axiovert 25 inverted microscope and an AxioCam HRc digital camera (Zeiss). Plaque areas were measured by using the NIH ImageJ software, and shown are the average plaque areas in calibrated units for each respective virus. The average area of plaques produced by the vC98A-1 was significantly smaller (≈50% for CKC and ≈25% for CEC) to the wild-type vRB-1B or revertant vC98A-1R (P < 0.01) by using Student's t tests. Shown below the histogram are representative plaques corresponding to the average plaque size of each virus.

Fig. 3.

qPCR assays of chicken blood. DNA was obtained from blood of chickens infected with parental vRB-1B, the C98A-1 mutant, or its revertant C98A-1R. Shown are MDV genome copies per million cells normalized to the chicken iNOS gene. Considerable numbers of chickens were terminated in the vRB-1B and revertant groups by day 36 p.i. because of development of multifocal lymphoma, and blood samples could be used for comparison with the vC98A-1 mutant only up to 29 days p.i. Levels of viral DNA were significantly different between the mutant (vC98A-1) and both the vRB-1B and vC98A-1R groups at 14, 22, and 29 days p.i. by using Student's t test (P < 0.01) as indicated with an asterisk. In the repeat experiment, a similar deficiency in the vC98A-1 group was seen (data not shown).

Fig. 4.

Tumorigenesis in viruses lacking USP activity is severely impaired. Marek's disease incidence was evaluated over the course of 92 days in two experiments (A and B) and is shown as a percentage of Marek's disease incidence for each group separated by lymphoma incidence. Lymphoma incidence show the percentage of chickens of each group that failed to develop lymphomas (0, white) or chickens in which less than (≤2, gray) or more than (>2, black) two sites of lymphoma manifested.

Fig. 5.

MDV^USP mRNA expression in MDV-transformed LCL. RT-PCR assays were performed on RNA obtained from the MDV-transformed cell line MSB-1 by using primers specific for the MDV^USP and gB genes as described in Materials and Methods. As negative controls, two cell lines transformed with REV (CU91 and CU205) were used, in addition to positive controls of MDV-infected CKC cultures. The CU210 cell line that was initially transformed with REV and then was superinfected and “supertransformed” with MDV was also used because tight transcriptional control of MDV genes was reported in this cell line (25). As an internal RNA control, GAPDH mRNA expression was also examined. One sample was used without addition of reverse transcriptase (−RT) to show the PCR products produced are specific for RNA and not DNA contamination.