The RING finger domain of Varicella-Zoster virus ORF61p has E3 ubiquitin ligase activity that is essential for efficient autoubiquitination and dispersion of Sp100-containing nuclear bodies

Abstract

Varicella zoster virus encodes an immediate-early (IE) protein termed ORF61p that is orthologous to the herpes simplex virus IE protein ICP0. Although these proteins share several functional properties, ORF61p does not fully substitute for ICP0. The greatest region of similarity between these proteins is a RING finger domain. We demonstrate that disruption of the ORF61p RING finger domain by amino acid substitution (Cys19Gly) alters ORF61p intranuclear distribution and abolishes ORF61p-mediated dispersion of Sp100-containing nuclear bodies. In addition, we demonstrate that an intact ORF61p RING finger domain is necessary for E3 ubiquitin ligase activity and is required for autoubiquitination and regulation of protein stability.

FIG. 1.

Redistribution of Sp100 in ORF61p WT or ORF61RFp-expressing cells. (A) Amino acid sequence of wild type ORF61p (NCBI protein database accession number NP_040183.1). Cys and His residues of the C₃HC₄ RING finger consensus sequence are indicated in red boldface. The asterisk identifies the Cys19Gly mutation that disrupts the RING finger domain to generate ORF61RFp. (B) 293A cells were mock transformed or transformed with pCK-flag-ORF61 or pCK-flag-ORF61RF. After 48 h, cells were harvested, and equal amounts of total protein were electrophoresed on NuPAGE 4 to 12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA) and then analyzed by Western blotting with antibodies specific for FLAG and actin. (C) MeWo cells grown on glass coverslips were transformed with pCK-flag-ORF61 or pCK-flag-ORF61RF. At 48 h posttransformation, cells were fixed, and the nuclear distribution of ORF61p (WT and RF) and Sp100 were monitored by indirect immunofluorescence microscopy using antibody specific for FLAG and Sp100, respectively. Nuclei were stained with Hoechst. Images were captured with a Zeiss Axiophot microscope using a ×100 objective lens and Open Lab software and analyzed by volume deconvolution. (D) For each sample, 50 cells were counted, and the number of ORF61p (WT and RF)-expressing cells was scored for Sp100-containing bodies. The results shown are from two independent experiments, each performed in duplicate.

FIG. 2.

Nuclear distribution of wild-type and RING finger mutant ORF61p. MeWo cells grown on glass coverslips were transformed with pCK-flag-ORF61 or pCK-flag-ORF61RF. At 38 h posttransformation, the cells were either treated with DMSO or 20 μM MG132. At 10 h posttreatment the cells were fixed, and the ORF61p (WT and RF) distribution was analyzed by indirect immunofluorescence microscopy with antibody specific for FLAG. Images were captured as described in Fig. 1. (A) Representative images for each protein and condition. (B) For each sample, 100 cells were counted, and the protein distribution was scored. The results shown are from three independent experiments.

FIG. 3.

Analysis of ORF61p ubiquitination. (A) 293A cells were mock transformed, transformed with pHA-Ub alone, or cotransformed with pHA-Ub and pCK-flag-ORF61 or pCK-flag-ORF61RF. After 48 h, cell lysates were prepared in lysis buffer and incubated overnight with EZview Red Anti-FLAG M2 affinity gel at 4°C. After incubation, immunocaptured (IC) proteins were eluted with FLAG peptide. Input and immunoprecipitated (IP) fractions were resolved on NuPAGE 4 to 12% Bis-Tris gradient gels and analyzed by Western blotting with antibody specific for FLAG and HA. (B) 293T cells were mock transformed or transformed with pCK-flag-ORF61 or pCK-flag-ORF61RF. After 48 h, cell lysates were prepared in lysis buffer and incubated for 1 h with EZview Red Anti-FLAG M2 affinity gel at 4°C. Bound (IC) proteins were either directly resolved on NuPAGE 4 to 12% Bis-Tris gradient gels (Invitrogen) or incubated with UBE1, His-UbcH3, UbcH5c, and ubiquitin (Boston Biochem) at 30°C for 2 h (+ Ub mix). Proteins were detected by Western blotting with antibody specific for FLAG.

FIG. 4.

Protein half-life analysis of wild type and RING finger mutant ORF61p. 293A cells were transformed with pCK-flag-ORF61 or pCK-flag-ORF61RF and after 24 h treated with 100 μg of cycloheximide (CHX)/ml. At various times posttreatment, cells were harvested directly by addition of Laemmli SDS-PAGE sample buffer, and equal volumes for each sample were run on NuPAGE 4 to 12% Bis-Tris gradient gels and analyzed by Western blotting with antibody specific for FLAG. Band intensity was quantified using ImageJ software.