Kaposi's Sarcoma-Associated Herpesvirus LANA Modulates the Stability of the E3 Ubiquitin Ligase RLIM

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. In a previous study, we have identified LANA-interacting proteins using a protein array screen. Here, we explore the effect of LANA on the stability and activity of RLIM (RING finger LIM-domain-interacting protein, encoded by the RNF12 gene), a novel LANA-interacting protein identified in that protein screen. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1, and the telomeric protein TRF1. Expression of LANA leads to downregulation of RLIM protein levels. This LANA-mediated RLIM degradation is blocked in the presence of the proteasome inhibitor, MG132. Therefore, the interaction between LANA and RLIM could be detected in coimmunoprecipitation assay only in the presence of MG132 to prevent RLIM degradation. A RING finger mutant RLIM is resistant to LANA-mediated degradation, suggesting that LANA promotes RLIM autoubiquitination. Interestingly, we found that LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM-mediated degradation of others, such as LHX3 and TRF1. We also show that transcription regulation by RLIM substrates is modulated by LANA. RLIM substrates are assembled into multiprotein transcription regulator complexes that regulate the expression of many cellular genes. Therefore, our study identified another way KSHV can modulate cellular gene expression.IMPORTANCE E3 ubiquitin ligases mark their substrates for degradation and therefore control the cellular abundance of their substrates. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1, and the telomeric protein TRF1. Here, we show that the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA protein enhances the ubiquitin ligase activity of RLIM, leading to enhanced RLIM autoubiquitination and degradation. Interestingly, LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM-mediated degradation of others, such as LHX3 and TRF1. In agreement with protein stability of RLIM substrates, we found that LANA modulates transcription by LHX3-LDB1 complex and suggest additional ways LANA can modulate cellular gene expression. Our study adds another way a viral protein can regulate cellular protein stability, by enhancing the autoubiquitination and degradation of an E3 ubiquitin ligase.

Keywords: E3 ubiquitin ligase; Kaposi's sarcoma; Kaposi's sarcoma-associated herpesvirus; LANA; LDB1; ORF73; RLIM.

FIG 1

LANA promotes RLIM degradation. (A) HEK293T cells were cotransfected with HA-RLIM and LANA expression vectors. Cell extracts were subjected to SDS-PAGE and Western blotting analysis with anti-RLIM (top), anti-LANA (middle), and anti β-actin (bottom) antibodies. (B and C) Same as in panel A, but cells were transfected with HA-RNF40 or FLAG-Itch, respectively. (D) Endogenous protein levels of RLIM were determined for cell extract from control SLK or rKSHV.219-infected SLK (SLK.219). (E) RLIM levels in three independent Western blotting analyses for SLK and SLK.219 were quantified by ImageJ and normalized to β-actin. RLIM mRNA (F) and LANA mRNA (G) levels in SLK and SLK.219 were determined by qRT-PCR. One-tailed t tests were performed for panels E through G, and significance was observed only in E and G (*, P ≤ 0.05; **, P ≤ 0.005). (H) HEK293T cells were transfected with LANA or empty vector and treated with 50 μg/ml cycloheximide for the indicated times. Endogenous RLIM levels were detected by Western blotting analysis. (I) RLIM and β-actin levels in Western blotting analyses of three independent cycloheximide experiments were quantified by ImageJ. Then, the amount of RLIM was divided by β-actin for each time point and presented in the graph. Results are presented as mean ± standard error of the mean (SEM).

FIG 2

LANA binds RLIM and promotes its degradation by the proteasome. (A) HEK293T cells were cotransfected with HA-RLIM and FLAG-LANA expression vectors. The cells were treated with 10 μM MG132 for 4 h or left untreated before cell extracts were prepared. Cell extracts were immunoprecipitated with anti-FLAG beads and subjected to SDS-PAGE and Western blotting analysis. Coimmunoprecipitated RLIM (top) and input RLIM (middle) were detected with anti-HA antibody. (B) BJAB and BC3 cells were treated with 10 μM MG132 for 4 h before cell lysates were prepared and immunoprecipitated with rat anti-LANA or normal rat IgG. (C) Schematic presentation of LANA mutants and summary of their interaction with RLIM. (D) HEK293T cells were cotransfected with HA-RLIM and LANA or LANA mutants, cells were treated with 10 μM MG132 for 4 h before cell extracts were prepared. Cell extracts were subjected to immunoprecipitation with anti-FLAG beads and subjected to SDS-PAGE and Western blotting analysis. (E) SLK and SLK.219 cells were treated with 10 μM MG132 for 4 h or left untreated before cells were fixed to a slide and immunostained with anti-RLIM (yellow), anti-LANA (purple) antibodies. Cell nucleus was stained with DAPI (blue).

FIG 3

LANA promotes RLIM ubiquitination. (A) HEK293T cells were cotransfected with HA-ubiquitin, FLAG-RLIM, and LANA expression vectors and kept untreated or treated with 10 μM MG132 for 2 h and 4 h before cell extracts were prepared. Cell extracts were immunoprecipitated with anti-FLAG beads, followed by high salt washes (1 M NaCl) and subjected to SDS-PAGE and Western blotting analysis. Immunoprecipitated ubiquitin (top) and RLIM (middle) were detected with anti-HA and anti-FLAG antibodies, respectively. LANA was detected in the input with anti-LANA antibody (bottom). (B) HEK293T cells were cotransfected with HA-RLIM or a RING finger mutant RLIM and FLAG-LANA expression vectors and kept untreated or treated with 10 μM MG132 for 4 h before cell extracts were prepared. Cell extracts were subjected to SDS-PAGE and Western blotting analysis with anti-HA (top), anti-LANA (middle), and anti β-actin (bottom) antibodies.

FIG 4

LANA modulates the stability of RLIM substrates. (A) Schematic illustration presenting the two alternative models for LANA effect on RLIM substrates. (B) HEK293T cells were transfected with HA-RLIM, HA-TRF1, and LANA expression vectors. Cell extracts were subjected to SDS-PAGE and Western blotting analysis. (C) Endogenous protein levels for TRF1 and RLIM were determined for cell extract from control TIME-Babe or TIME-LANA. (D) RNA levels of TRF1, RLIM, and LANA were determined by qRT-PCR. One-tailed t tests were performed, and significance was observed only in LANA (*, P ≤ 0.05). (E) HEK293T cells were transfected with HA-RLIM, Myc-LHX3, and LANA expression vectors, and cell extracts were subjected to SDS-PAGE and Western blotting analysis. Anti-Myc antibody was used to detect the Myc tag LHX3. (F) HEK293T cells were transfected with HA-RLIM, LANA, and FLAG-LDB1 or FLAG-LMO2 expression vectors. Cell extracts were subjected to SDS-PAGE and Western blot analysis with anti-FLAG (top) and anti-HA (bottom) antibodies. (G) Endogenous protein levels for LDB1 and RLIM were determined for cell extract from control TIME-Babe or TIME-LANA.

FIG 5

LANA represses LHX3-LDB1-dependent transcription. CHO (A) and HEK293T (B) cells were cotransfected with thyroid-stimulating hormone beta-subunit (TSH-b) promoter fused to luciferase reporter gene and PITX1, LHX3, LANA, RLIM, and LDB1 expression vectors. (C) CHO and HEK293T cells extracts were subjected to SDS-PAGE and Western blotting analysis to determined endogenous protein levels for LDB1 (top) and β-actin (bottom). (D and E) Same as in panel A, CHO (D) and HEK293T (E) cells were cotransfected but with the addition of RING finger mutant RLIM (RLIM RINGm) expression vector. Data for luciferase assay are reported as the relative luciferase activity (RLU), and results represent the mean ± SEM.

FIG 6

Schematic model for the effects of LANA and RLIM on the ability of LHX3 and LDB1 to activate transcription. A schematic model presenting the transcription activity dependent on protein complexes of LHX3 and LDB1 (A), in cells expressing LANA (B), RLIM (C), LANA and RLIM (D), or LANA, RLIM, and overexpressed LDB1 (E).