The Rad6/18 ubiquitin complex interacts with the Epstein-Barr virus deubiquitinating enzyme, BPLF1, and contributes to virus infectivity

Abstract

Epstein-Barr virus (EBV) encodes BPLF1, a lytic cycle protein with deubiquitinating activity that is contained in its N-terminal domain and conserved across the Herpesviridae. EBV replication is associated with cellular DNA replication and repair factors, and initiation of EBV lytic replication induces a DNA damage response, which can be regulated at least in part by BPLF1. The cellular DNA repair pathway, translesion synthesis (TLS), is disrupted by BPLF1, which deubiquitinates the DNA processivity factor, PCNA, and inhibits the recruitment of the TLS polymerase, polymerase eta (Pol eta), after damage to DNA by UV irradiation. Here we showed that the E3 ubiquitin ligase, which activates TLS repair by monoubiquitination of PCNA, is also affected by BPLF1 deubiquitinating activity. First, BPLF1 interacts directly with Rad18, and overexpression of BPLF1 results in increased levels of the Rad18 protein, suggesting that it stabilizes Rad18. Next, expression of functionally active BPLF1 caused relocalization of Rad18 into nuclear foci, which is consistent with sites of cellular DNA replication that occur during S phase. Also, levels of Rad18 remain constant during lytic reactivation of wild-type virus, but reactivation of BPLF1 knockout virus resulted in decreased levels of Rad18. Finally, the contribution of Rad18 levels to infectious virus production was examined with small interfering RNA (siRNA) targeting Rad18. Results demonstrated that reducing levels of Rad18 decreased production of infectious virus, and infectious titers of BPLF1 knockout virus were partially restored by overexpression of Rad18. Thus, BPLF1 interacts with and maintains Rad18 at high levels during lytic replication, which assists in production of infectious virus.

Importance: Characterization of EBV BPLF1's deubiquitinating activity and identification of its targets and subsequent functional effects remain little studied. All members of the Herpesviridae contain BPLF1 homologs with conserved enzymatic activity, and findings discovered with EBV BPLF1 are likely applicable to other members of the family. Discovery of new targets of BPLF1 will point to cellular pathways and viral processes regulated by the enzymatic activity of the EBV-encoded deubiquitinating enzyme. Here we determined the importance of the cellular ubiquitin ligase Rad18 in these processes and how it is affected by BPLF1. Our findings demonstrate that EBV can co-opt Rad18 as a novel accessory factor in the production of infectious virus.

FIG 1

Rad18 interacts with BPLF1 in vivo. (A) H1299 cells were transfected with FLAG-tagged BPLF1 1-246. Lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting with FLAG, Rad18, and PCNA antibodies. Whole-cell lysates were also probed to indicate input levels. (B) Reverse immunoprecipitations were performed to confirm BPLF1 interaction with Rad18. H1299 cells were cotransfected with BPLF1 1-246 and Myc-tagged Rad18. Immunoprecipitations were performed with Myc antibody, followed by immunoblot analysis with Myc, FLAG, and HA antibodies. Whole-cell lysates were also probed.

FIG 2

Rad18 interacts with BPLF1 but does not compete with PCNA for a common binding site on BPLF1. (A) BPLF1 interacts with Rad18 in vitro. BPLF1 and Rad6/Rad18 protein complex expressed and purified from E. coli were incubated together at equimolar concentrations (lanes 1 to 3) at room temperature for 1 h and then immunoprecipitated with anti-BPLF1 antibody, and Western blots were probed with Rad18, PCNA, and BPLF1 antibodies. Results showed that BPLF1 interacts with Rad18 independently of PCNA. Additionally, the BPLF1 protein (10 nM) was incubated with either Rad18 (10 nM) and 10-fold excess of PCNA (100 nM) (lane 4) or PCNA (10 nM) and RAD18 (100 nM) (lane 5) and subjected to immunoprecipitation with BPLF1 antibody. (B) PCNA and Rad18 do not compete for a common binding site on BPLF1. A semi-in vivo assay was used. FLAG-BPLF1 was expressed in H1299 cells, and whole-cell lysates were incubated with increasing concentrations of purified PCNA and Rad18 at room temperature for 1 h. Reaction mixtures were immunoprecipitated with FLAG antibody and probed for PCNA, Rad18, and BPLF1. Results indicate no competition between Rad18 and PCNA for BPLF1 binding.

FIG 3

BPLF1 deubiquitinates the Rad6/Rad18 complex. (A) BPLF1 deubiquitinates Rad6/18 in vitro. Ube1 was incubated with Rad6/Rad18 and FLAG-ubiquitin in the presence or absence of BPLF1. Samples were run on a 12% SDS-PAGE gel, immunoblotted, and probed with FLAG, Rad18, and BPLF1 antibody. BPLF1 specifically deubiquitinates Rad6 but not Ube1. Slight deubiquitination of Rad18 was also observed. Input levels are shown at the bottom. (B) Rad18 is likely deubiquitinated by BPLF1 in vivo. 293EBV+ cells were transfected with BPLF1 1-246 and BZLF1 (to induce lytic replication) where indicated. The upper band in the Rad18 panel is consistent with the monoubiquitinated form of Rad18 and appears to be reduced in the presence of BPLF1. BPLF1 and GAPDH loading controls are shown in the middle and bottom panels.

FIG 4

Expression of BPLF1 increases Rad18 focus formation. Two separate fields of view are shown for each condition (left panels versus right panels [A-C]). (A) H1299 cells transfected with YFP-Rad18 are homogeneously distributed and are localized primarily in the nucleus. (B) H1299 cells coexpressing the inactive BPLF1 C61S and YFP-Rad18 do not affect focus formation. (C) H1299 cells coexpressing FLAG-BPLF1 and YFP-Rad18 show increased nuclear focus formation of Rad18. (D) Quantification of percentages of cells with focus formation in panels A, B, and C. One hundred cells were counted for each sample.

FIG 5

Endogenous BPLF1 expression induces Rad18 focus formation. (A) 293EBV+ WT and 293EBV+ BPLF1 knockout cell lines were transfected with YFP-Rad18 (upper panels), additionally transfected with BZLF1 to induce lytic replication (middle panels), and induced and supplied with BPLF1 exogenously (lower panels). Induction of WT virus produced Rad18 foci but not BPLF1 knockout virus. Cell lines containing the EBV genomes are GFP tagged. EA-D (red) is an early lytic gene and demonstrates viral reactivation. (B) Quantitation of cells containing Rad18 foci. Twenty cells under each condition were examined for the presence of Rad18 foci.

FIG 6

BPLF1 expression increases Rad18 protein levels. (A) 293EBV+ cells were transfected with BPLF1 1-246, and the lytic cycle was induced with BZLF1. Immunoblotting revealed a substantial increase in Rad18 protein levels when BPLF1 was expressed. Rad6 levels remained largely unchanged. EA-D is an EBV early protein that indicates induction of the lytic program. (B) 293T cells were transfected with functional (BPLF1 1-246) or nonfunctional (BPLF1 C61S) BPLF1. Rad18 levels were increased only in the presence of functional BPLF1. (C) Induction of BPLF1 knockout virus results in less Rad18 protein levels than with the WT. WT and BPLF1 knockout viruses were induced into the lytic replication cycle by transfection of BZLF1. Levels of Rad18 were normalized to GAPDH protein levels.

FIG 7

Rad18 levels contribute to viral infectivity. Knockdown of Rad18 levels with siRNA reduces viral infectivity (A to C). (A) Extracellular genome copies. 293EBV+ cells were treated with siRNA against Rad18 and induced with BZLF1 for 48 h. DNA was extracted from supernatant fluids, and genome copies were detected with quantitative PCR (qPCR) targeting the BamHI repeat region. (B) Viral infectivity. The same supernatant fluids collected for assay of extracellular genome copy number were assayed for infectious titer on Raji cells. Infectivity was determined with flow cytometry 48 and 72 h after infection. (C) Immunoblots showing knockdown of Rad18. (D) Viral infectivity of BPLF1 knockout virus is partially restored by overexpression of Rad18. BPLF1 knockout virus was induced with Z and assayed for infectivity on Raji cells. Rad18 and BPLF1 were overexpressed as indicated. Experiments were performed in triplicate, and error bars represent the standard errors of means.