Myc is required for the maintenance of Kaposi's sarcoma-associated herpesvirus latency

Abstract

Myc is deregulated by Kaposi's sarcoma-associated herpesvirus (KSHV) latent proteins, but its role in KSHV latency is not clear. We found that Myc knockdown with RNA interference (RNAi) induced KSHV reactivation and increased the protein and mRNA levels of RTA, a key viral regulator of KSHV reactivation. Myc knockdown increased, whereas Myc overexpression inhibited, RTA promoter activity. KSHV reactivation and the activation of the RTA promoter induced by Myc depletion were inhibited by c-Jun N-terminal kinase (JNK) and p38 inhibitors but not by a MEK1 inhibitor. Myc knockdown inhibited primary effusion lymphoma (PEL) cell proliferation through inducing apoptosis and G(1) cell cycle arrest. Thus, Myc may be a key cellular node coupling cellular transformation and KSHV latency.

FIG. 1.

Myc knockdown induces KSHV reactivation. (A) BC-3-G cells that were mock transduced or transduced with the indicated shRNA vectors were imaged with fluorescence microscopy for enhanced green fluorescence protein (EGFP) expression at day 3 posttransduction. Magnification, ×200. (B) BC-3 or BCBL-1 cells were mock transduced or transduced with the indicated shRNA vectors. At day 3 posttransduction, whole-cell lysates were collected and analyzed for KSHV K8 and/or Myc expression by Western blotting. An actin immunoblot is shown as a loading control. (C) BC-3 cells were cotransfected with the indicated shRNA plasmids. At day 3 posttransduction, whole-cell lysates were collected and analyzed for KSHV RTA expression by Western blotting. A tubulin immunoblot is shown as a loading control. Data for all panels are representative of three independent experiments.

FIG. 2.

Myc represses RTA transcription. (A) BC-3 cells were transduced with the indicated shRNA vectors. At day 2 posttransduction, the relative RTA mRNA levels were measured by reverse transcription-quantitative PCR (RT-Q-PCR), normalized to the actin mRNA levels, and displayed as fold induction over the shCtrl control. (B) 293T cells were cotransfected with pKRp (RTA promoter luciferase reporter plasmid) or pGL2 (control reporter plasmid) plus the indicated shRNA plasmids. At 48 h posttransfection, the luciferase activities were measured and are presented as relative fold induction over the luciferase activity of the pGL2-plus-shCtrl sample. (C) 293T cells were cotransfected with the indicated luciferase reporter and expression plasmids. At 48 h posttransfection, the luciferase activities were determined and are presented as relative fold induction over the luciferase activity of the pGL2-plus-Ctrl sample. (D) 293T cells were cotransfected with pPAN-Luc (PAN promoter luciferase reporter plasmid) and the indicated expression plasmids. At 48 h posttransfection, the luciferase activities were determined and are presented as relative fold induction over the luciferase activity of the sample without Myc and RTA. Data are the means ± standard deviations from three independent experiments.

FIG. 3.

Requirement of JNK and p38 signaling for KSHV reactivation induced by Myc knockdown. (A) BC-3 cells were transfected with shCtrl or shMyc-1. At day 2 posttransfection, cells were treated with 20 μM PD 98059, SB 203580, SP 600125, or the dimethyl sulfoxide (DMSO) vehicle control. At day 3 posttreatment, cells were collected and analyzed by Western blotting. A tubulin immunoblot is shown as a loading control. Data are representative of two independent experiments. (B) 293T cells were cotransfected with pKRp and the shCtrl or shMyc-1 plasmid. At 12 h posttransfection, cells were treated with 20 μM PD 98059, SB 203580, SP 600125, or the DMSO vehicle control. At 60 h posttransfection, the luciferase activities were measured and are presented as relative fold induction over the luciferase activity of the shCtrl-plus-DMSO sample. Data are the means ± standard deviations from three independent experiments.

FIG. 4.

Myc knockdown induces G₁ cell cycle arrest and apoptosis. (A) BC-3 cells were transduced with the shCtrl or shMyc-1 lentiviral vector. Cell numbers were determined by the trypan blue exclusion assay at day 4 posttransduction and are displayed as relative cell densities normalized to the number of shCtrl-transduced cells. Data are the means ± standard deviations from three independent experiments. (B) Flow cytometry evaluation of the cell cycle in shCtrl- and shMyc-1-transduced BC-3 cells at day 4 posttransduction. The percentage of cells in each phase of the cell cycle was determined by analysis performed with Modfit LT software. Data are representative of two independent experiments.