Epstein-Barr virus-induced miR-155 attenuates NF-kappaB signaling and stabilizes latent virus persistence

Abstract

MicroRNAs have been implicated in the modulation of gene expression programs important for normal and cancer cell development. miR-155 is known to play a role in B-cell development and is upregulated in various B-cell lymphomas, including several that are latently infected with Epstein-Barr virus (EBV). We show here that EBV infection of primary human B lymphocytes leads to the sustained elevation of miR-155 and its precursor RNA, BIC. The EBV-encoded latency membrane protein 1 (LMP1) can partially reconstitute BIC activation in B lymphocytes but not in epithelial cell cultures. LMP1 is a potent activator of NF-kappaB signaling pathways and is essential for EBV immortalization of B lymphocytes. An inhibitor to miR-155 further stimulated NF-kappaB responsive gene transcription, and IKKepsilon was identified as a potential target of miR-155 translational repression. Remarkably, miR-155 inhibitor reduced EBNA1 mRNA and the EBV copy number in latently infected cells. This suggests that miR-155 contributes to EBV immortalization by modulation of NF-kappaB signaling and the suppression of host innate immunity to latent viral infection.

FIG. 1.

miR-155 and BIC are induced by EBV immortalization of primary B lymphocytes. (A) Total RNA was isolated from human peripheral blood cells from seven different donors at 10 days postinfection with EBV (EBV), at 10 days after mock infection (Mock), or immediately prior to EBV infection (Pre). RNA was analyzed for miRNA expression by using differential hybridization. miRNA expression was grouped by hierarchical cluster analysis with RNA samples in the vertical axis and by relevant miRNAs in the horizontal axis. (B) RNA was isolated from preinfected peripheral blood cell samples or from peripheral blood cell samples (1 to 7) at 10 days postinfection or at 10 days after mock infection and assayed by RT-PCR for the miR-155 precursor BIC RNA relative to cellular actin RNA. (C) Northern blot analysis of total RNA isolated from samples 4, 5, and 6 from preinfection (D0), mock-infected (−), or EBV (+)-infected peripheral blood samples (10 days postinfection) and analyzed with a probe specific for miR-155 (top panel) or control U6 snRNA (lower panel). (D) BIC RNA relative to actin RNA was measured by RT-PCR at various days (as indicated) after EBV infection or after mock infection of peripheral blood cells. (E) EBV-encoded LMP1 and EBNA2, along with BIC RNA, were measured at 10 days posttransfection relative to actin RNA using RT-PCR for EBV-infected samples from donors 4, 5, and 6.

FIG. 2.

BIC RNA levels are highest in EBV-positive LCL cells. (A) BIC RNA levels were assayed by RT-PCR and normalized to actin RNA levels for various cell lines (DG75 is an EBV-negative BL cell line, Raji is EBV-positive BL cell line, LCL is an EBV-positive lymphoblastoid cell line, 293T is an EBV-negative HEK cell line, ZKO is a Zta knockout EBV bacmid-positive 293 cell line, and D98 is an EBV P3HR1 strain-positive HeLa cell line). (B and C) Same as in panel A, except that LMP1 (B) and EBNA-2 (C) mRNA were quantified relative to actin for each cell line.

FIG. 3.

B-cell-specific induction of BIC RNA by LMP1. (A to C) EBV-negative BL cell DG75 (5 × 10⁶ cells) was transfected with 5 μg of expression vectors for LMP1, EBNA2, LMP1+EBNA2, or control vector (mock) and compared to Raji cell RNA for expression of BIC (A), EBNA-2 (B), or LMP1 (C) relative to actin RNA. (D to F) EBV-negative 293T cells were transfected and analyzed as described for panels A to C.

FIG. 4.

miR-155 modulates NF-κB signaling in B lymphocytes. (A) miR-155 inhibitor or control inhibitor (0 to 100 μM) were assayed for their ability to relieve repression of the miR-155 target sequence in LCL cells (3 × 10⁶) using a luciferase indicator plasmid (2 μg). (B) LCL cells were transfected with 5x κB-L reporter plasmid and either control or miR-155 inhibitor and assayed for luciferase activity. (C) EBV-negative DG75 BL cells were transfected with pGL3-Basic or 5x κB-L reporter plasmids and either miR-155 mimic or control and then assayed for luciferase activity. (D) Same as in panel C, except in 293T cells.

FIG. 5.

Suppression of the IKKɛ pathway by miR-155. (A) Western blot analysis of IKKɛ protein (top panel) or PCNA protein control (lower panel) in either DG75 cells (left two lanes) transfected with miR-155 mimic (+) or control (−), or EBV-positive LCL cells (right two lanes) transfected with miR-155 inhibitor (+) or control (−). (B) IKKɛ, BCL2, or CCL5 mRNA was measured relative to actin mRNA in LCL cells transfected with miR-155 inhibitor (▪) or control (□). (C) Same as in panel B, except in Raji cells.

FIG. 6.

miR-155 stabilizes EBV genome copy number during latent infection. (A) EBV DNA copy number was assayed by real-time PCR of EBV dyad symmetry (DS) DNA relative to cellular actin in Raji cells transfected with miR-155 inhibitor or control. (B) Same as in panel A, except in LCL cells. (C) Raji cells were transfected with miR-155 inhibitor or control and then assayed for EBNA1 mRNA expression relative to actin using RT-PCR. (D) Same as in panel C, except in LCL cells. (E) Southern blot analysis of EBV genomic DNA (upper panel) and cellular telomere repeat DNA (Telo, lower panel) in control (Ctrl) or miR-155 inhibitor-transfected Raji cells. (F) Summary of genetic interactions of miR-155 relevant to EBV immortalization and stable latent infection. EBV LMP1 induces BIC/miR-155, which in turn attenuates NF-κB signaling and IKKɛ protein expression. miR-155 also contributes to EBV genome maintenance, in part by stimulating EBNA1 mRNA expression.