An Internally Translated MAVS Variant Exposes Its Amino-terminal TRAF-Binding Motifs to Deregulate Interferon Induction

<div><p>Activation of pattern recognition receptors and proper regulation of downstream signaling are crucial for host innate immune response. Upon infection, the NF-κB and interferon regulatory factors (IRF) are often simultaneously activated to defeat invading pathogens. Mechanisms concerning differential activation of NF-κB and IRF are not well understood. Here we report that a MAVS variant inhibits interferon (IFN) induction, while enabling NF-κB activation. Employing herpesviral proteins that selectively activate NF-κB signaling, we discovered that a MAVS variant of ~50 kDa, thus designated MAVS50, was produced from internal translation initiation. MAVS50 preferentially interacts with TRAF2 and TRAF6, and activates NF-κB. By contrast, MAVS50 inhibits the IRF activation and suppresses IFN induction. Biochemical analysis showed that MAVS50, exposing a degenerate TRAF-binding motif within its N-terminus, effectively competed with full-length MAVS for recruiting TRAF2 and TRAF6. Ablation of the TRAF-binding motif of MAVS50 impaired its inhibitory effect on IRF activation and IFN induction. These results collectively identify a new means by which signaling events is differentially regulated via exposing key internally embedded interaction motifs, implying a more ubiquitous regulatory role of truncated proteins arose from internal translation and other related mechanisms.</p></div

Characterize the roles of MAVS50 in RIG-I-dependent signaling.

<p>(A and B) 293T cells were transfected with an NF-κB (A) or IFN-β (B) reporter cocktail and increasing amount of MAVS wild-type (WT), MAVS70 or MAVS50. Reporter activation was determined by luciferase assay at 30 hours post-transfection. (C) 293T cells were transfected with plasmids containing indicated genes. At 48 hours post-transfection, IKKβ kinase was precipitated and analyzed by in vitro kinase assay and immunoblotting with anti-IKKβ. Whole cell lysates were analyzed with anti-Flag (MAVS) and anti-GST (RIG-I-N). (D) 293T cells were transfected with plasmids containing Flag-MAVS70 and Flag-MAVS50. MAVS70 and MAVS50 were purified by affinity chromatography, eluted and analyzed by gel filtration chromatography with Superdex 200. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag antibody. V₀, void volume; numbers at the top indicate molecular weight in kDa. (E and F) MAVS in 293T cells was depleted with shRNA and analyzed by immunoblotting (E) and MAVS expression was “reconstituted” with lentivirus containing MAVS wild-type (WT), MAVS70 or MAVS50. Whole cell lysates were analyzed with anti-V5 antibody (F). (G) MAVS knockdown 293T cells “reconstituted” with control lentivirus (Vec) or lentivirus containing MAVS wild-type (WT), MAVS70 (70) or MAVS50 (50) as shown in (F), were mock- or infected with Sendai virus (SeV, 100 HAU/ml) for 8 hours, WCLs were prepared and analyzed by immunoblotting with indicated antibodies. (H) Infection of “reconstituted” 293T cells as described in (G). Mitochondrion-enriched fraction was obtained and analyzed by immunoblotting with indicated antibodies. (I and J) MAVS knockdown 293T cells, “reconstituted” with MAVS expression as described in (F), were infected with SeV (100 HA unit/ml) for 8 hours, RNA was extracted, cDNA was prepared and real-time PCR with primers specific for <i>hIFNb</i> and <i>CCL5</i> were performed (I). Supernatants were collected and hIFNβ and hCCL5 were determined by ELISA (J).</p

MAVS50 interacts with MAVS70.

<p>(A) 293T cells were transfected with plasmids containing MAVS wild-type, MAVS70 or MAVS50. Whole cell lysates (WCLs) were precipitated with anti-Flag (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (B) 293T-Rex/MAVS50-Flag cell line was induced with doxycycline (100 ng/ml) for 24 hours. WCLs were precipitated with anti-Flag agarose (MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (C) 293T cells were transfected with a plasmid containing MAVS50-Flag, with or without a plasmid containing MAVS70-V5. MAVS50 was purified by affinity chromatography, eluted and analyzed by gel filtration chromatography. Fractions (30 μl) were analyzed by immunoblotting with anti-Flag and anti-V5 antibodies. (D) Whole cell lysates of 293T, HeLa and THP-1 macrophage were analyzed by gel filtration chromatography. Fractions (50 μl) were analyzed by immunoblotting with anti-MAVS antibody. For C and D, V₀, void volume; numbers indicate molecule weight in kDa.</p

The N-terminal TRAF2-binding motif is critical for MAVS50 to inhibit IFN induction.

<p>(A) Diagram of the TRAF2-binding motif (T2BM) and TRAF6-binding motif (T6BM) in MAVS50, in relation to MAVS70. (B and C) 293T cells were transfected with plasmids containing Flag-TRAF2 (B) or Flag-TRAF6 (C) and plasmids containing MAVS50 wild-type (WT), mutant of TRAF2-binding (M2) or TRAF6-binding (M6) or both TRAF2- and TRAF6-binding (M2,6). Whole cell lysates (WCLs) were prepared at 30 hours post-transfection and precipitated with anti-Flag agarose or anti-HA agarose (as negative control). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (D) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50 wild-type (WT) or MAVS50 M2,6 mutant. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. <i>**p<0</i>.<i>01</i>; <i>***p<0</i>.<i>005</i>. (E) 293T cells were infected with control lentivirus (CTL) or lentivirus containing MAVS50 wild-type or MAVS50 M2,6 mutant. At 48 hours, cells were infected with SeV (100 HA unit/ml) for 8 hours. RNA was extracted and cDNA were prepared for real-time PCR analysis with primers specific for <i>IFNβ</i> and <i>ISG56</i>. (F and G) 293T cells were transfected with an empty plasmid (Vector) or a plasmid containing MAVS50 or MAVS50 M2,6 mutant. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (F) and virus in the supernatant was determined by plaque assay (G).</p

Identification of the MAVS50 variant.

<p>(A) 293T cells were infected with SeV (100 HA unit/ml) for 2 hours or transfected with a plasmid containing murine γHV68 vGAT for 24 hours. Whole cell lysates (WCL) were prepared in Triton X-100 buffer to separate into soluble and insoluble (pellet) fractions, which were analyzed by immunoblotting with indicated antibodies. (B) Diagram of the mRNA of the full-length MAVS (or MAVS70) and MAVS50. (C) WCLs of indicated amount were analyzed with antibodies against the first 135 amino acids (αMAVS^1-135) or an internal sequence encompassing amino acids 150–250 (αMAVS^150-250). (D) The expression of MAVS, carrying a N-terminal Flag tag and a C-terminal HA tag, in 293T cells was analyzed by immunoblotting with anti-Flag and anti-HA antibodies, along with endogenous MAVS (left panel). (E) 293T cells were transfected with plasmids containing wild-type MAVS and indicated mutants. WCLs were analyzed by immunoblotting with anti-MAVS antibody (αMAVS^150-250). Δ, deletion. (F) WCLs of indicated cells were analyzed by immunoblotting with anti-MAVS (αMAVS^150-250) and anti-β-actin. Note, antibody against human MAVS does not react with murine MAVS in NIH 3T3 cells. (G and H) 293T cells were infected with Sendai virus (SeV, 100 HA Unit/ml) (G) and HSV-1 (MOI = 5) (H) and cells were harvested at indicated time points. WCLs were analyzed by immunoblotting with antibody against MAVS.</p

MAVS50 targets TRAF molecules to inhibit MAVS70-mediated IFN induction.

<p>(A) Diagram of key signaling molecules of the RIG-I-dependent IFN induction. (B and C) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid containing RIG-I-N (B) or TBK-1 (C), and increasing amount of plasmid containing MAVS50. The IFN-β promoter activity was determined by luciferase assay at 30 hours post-transfection. <i>**p<0</i>.<i>01</i>; <i>***p<0</i>.<i>005</i>. (D) 293T cells were transfected with plasmids containing MAVS wild-type (WT) or MAVS50 and a plasmid containing TRAF6. Whole cell lysates (WCL) were precipitated with anti-Flag (TRAF6). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (E) 293T cells, depleted with endogenous MAVS and “reconstituted” with MAVS70 or MAVS50, were precipitated with anti-V5 (MAVS70 or MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. (F) MAVS knockdown cells “reconstituted” with MAVS70-V5 were transfected with increasing amount of MAVS50-Flag. At 30 hours post-transfection, cells were infected with Sendai virus (SeV, 200 HA unit/ml) for two hours. WCLs were prepared and precipitated with anti-V5 (MAVS70) antibody. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. V, vector.</p

MAVS50 inhibits MAVS70-dependent IFN induction.

<p>(A and B) 293T cells were transfected with an IFN-β (A) or NF-κB reporter cocktail (B), a plasmid containing MAVS70 and increasing amount of a plasmid containing MAVS50. The promoter activity of IFN-β and NF-κB was determined by luciferase assay at 30 hours post-transfection. <i>**p<0</i>.<i>01</i>; <i>***p<0</i>.<i>005</i>. (C and D) 293T cells were infected with control (CTL) lentivirus or lentivirus containing MAVS50. Whole cell lysates were analyzed with indicated antibodies (C). Stable 293T cells were infected with SeV (100 HA unit/ml) for 8 hours and RNA was extracted. cDNA was prepared and analyzed by real-time PCR with primers specific for <i>IFNβ</i> and <i>ISG56</i> (D). (E and F) 293T cells were transfected with vector or plasmids containing MAVS wild-type (WT), MAVS70 or MAVS50. At 24 hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-infection (E) and VSV in the supernatant was determined by plaque assay (F).</p

PtdIns(3)P-bound UVRAG coordinates Golgi-ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin&nbsp;1 complex.

Endoplasmic reticulum (ER)-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-beclin-1-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis