Respiratory syncytial virus NS1 protein degrades STAT2 by using the Elongin-Cullin E3 ligase

Abstract

Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants. RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.

FIG. 1.

NS1 interacts with elongin C and Cul2. (A and B) CLUSTALW protein alignments of RSV NS1 protein and elongin C-interacting proteins (VHL and SOCS1 to -3) (A) and (B) Cul2-interacting proteins (VHL, FEM1B, LLR1). Identical (black) and similar (gray) amino acids are highlighted. (C) MYC-elongin C expressed in WCLs from 293T cells (top panel) were incubated with HIS-NS1 or HIS-Zap70 (control) precoupled to nickel-NTA beads. Membranes were immunoblotted (IB) with anti-MYC (left side, upper panel) to detect elongin C or with anti-HIS to detect HIS-Zap70 or HIS-NS1 protein (left side, lower panels). Association between MYC-elongin C and HIS-NS1 or HIS-NS2 was analyzed as above (right panels). (D) HA-Cul2 expressed in WCLs from 293T cells (top panel) was incubated with HIS-NS1 or HIS-SOCS2 (control) precoupled to nickel-NTA beads. Membranes were IB with anti-HA (left side, upper panel) to detect Cul2 or with anti-HIS to detect HIS-SOCS2 or HIS-NS1 (left side, lower panels). Association between HA-Cul2 and HIS-NS1 or HIS-NS2 was analyzed as above (right panels).

FIG. 2.

Effects of NS1 and NS2 on STAT1 and STAT2 in 293T cells. (A) Ev, or NS1 or NS2 cDNA, was transfected into 293T cells. Cells were treated with proteasome inhibitors (MG132 and LLnL) 4 h prior to lysis to confirm NS1 expression (left panel) or lysed in 8 M urea to analyze NS2 expression (right panel). (B and C) Cells were transfected with cDNA encoding FLAG-STAT1 (B) or STAT2 (C) in the presence of NS1, NS2, or both NS1 and NS2. Membranes were probed with anti-FLAG to detect STAT1 or anti-STAT2 (upper panels) and reprobed with anti-γ-tubulin to confirm equal protein loading in all lanes (lower panels). (D) Loss of STAT2 protein by NS1 and NS2 can be prevented by blocking proteasomal activity. 293T cells were transfected with vectors expressing STAT2 in the absence and presence of NS1/2. Cells were treated with and without proteasome inhibitors (MG132 and LLnL) 3 h before lysing. STAT2 levels were analyzed by immunoblotting membranes with anti-STAT2 (upper panels). Membranes were reprobed with anti-γ-tubulin (lower panels).

FIG. 3.

NS1/2-dependent loss of STAT2 is inhibited by a siRNA directed against E3 ligase components. (A) Effect of siRNA on E3 ligase mRNA levels in 293T cells. 293T cells were transfected with a siRNA empty vector (Ev) control or with siRNA directed against Cul2 (Cul2-1 and Cul2-2), Cul5 (Cul5-1 and Cul5-2), Rbx1 (Rbx1-1 and Rbx1-2), or Rbx2 (Rbx2-1 and Rbx2-2). At 48 h after transfection, RNA was isolated and RT-PCR performed using primer pairs specific to each E3 ligase component (upper panels). Control reactions with β-actin primers confirmed equal quantities of RNA in each reaction (lower panels). (B) Loss of STAT2 by NS1/2 can be inhibited by downregulating Cul and Rbx expression. 293T cells were transfected with a siRNA empty vector (Ev) control or with a siRNA directed against Cul2 (Cul2-1 and Cul2-2), Cul5 (Cul5-1 and Cul5-2), Rbx1 (Rbx1-1 and Rbx1-2) or Rbx2 (Rbx2-1 and Rbx2-2) in the presence of STAT2 and/or NS1/2. Lysates were analyzed for STAT2 expression by immunoblotting with a STAT2 antibody (upper panel) and were reprobed with an antibody against γ-tubulin (lower panel).

FIG. 4.

Loss of STAT2 expression in HEp-2 cells infected with RSV. HEp-2 cells were either mock infected or infected with RSV at an MOI of 15. After 24 h, cells were treated with and without IFN-β (1,000 U/ml for 30 min) both in the presence and in the absence of proteasome inhibitors (MG132 and LLnL), which were added 3 h prior to lysis. Whole-cell lysates were analyzed for STAT1, STAT2, phosphorylated STAT1 (pSTAT1), phosphorylated STAT2 (pSTAT2), or γ-tubulin.

FIG. 5.

Loss of STAT2 protein in RSV-infected HEp-2 cells is rescued with a siRNA against Rbx1 or Cul2. HEp-2 cells were transfected with a siRNA empty vector (Ev) or a siRNA directed against Cul2 (Cul2-1 and Cul2-2), Cul5 (Cul5-1 and Cul5-2), Rbx1 (Rbx1-1 and Rbx1-2), or Rbx2 (Rbx2-1 and Rbx2-2). A Lipofectamine transfection control was also included (Lc). Cells were then either mock infected or infected with RSV at an MOI of 15. Whole-cell lysates were analyzed for STAT2 (upper panels), STAT1 (middle panels), or γ- tubulin (lower panels) expression.

FIG. 6.

STAT2 is modified by ubiquitin. 293T cells were transfected with vectors expressing NS1/2, STAT2, and HA-ubiquitin both in the presence and in the absence of exogenous E3 ligase components elongin BC, Cul2, and Rbx1. Cells were treated with proteasome inhibitors (MG132 and LLnL), which were added 3 h prior to lysis. Whole-cell lysates were analyzed for STAT2 (upper panel) or ubiquitin (lower panel).