A Viral Protein Restricts Drosophila RNAi Immunity by Regulating Argonaute Activity and Stability

Abstract

The dicistrovirus, Cricket paralysis virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, we combined structural, biochemical, and virological approaches to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2 binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and virus replication. Our study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.

Keywords: Ago-2; Ago-2 degradation; CrPV; E3 ligase; RNAi; RNAi suppressor; antiviral immunity; insects.

Fig 1:. Overview of CrPV-1A structure

(A, B) Side and top view of CrPV-1A monomer (C) Representation of the surface electrostatic potential in the CrPV-1A with negatively charged regions colored in red and corresponding positively charged regions is in blue. (D) Two adjacent monomers [monomer 1(cyan) and monomer 2(orange)] create two contact interfaces. Interface I is formed by hydrogen bonding between pairs of glutamic acids (Glu55) and tyrosine residues (Tyr57) on antiparallel beta strands. Interface II forms due to crystal contacts and is driven by hydrogen bonding between arginine 71 (Arg71) and glutamate 74 (Glu74) and electrostatic interactions (ES) between lysine 77 (Lys77) and glutamate 92 (Glu92). See also Fig S1 and Table S1.

Fig 2:. A flexible loop in CrPV-1A inhibits RNAi response in Drosophila cells

(A) Schematic of a dual-luciferase reporter assay to screen for RNAi suppression (B) The CrPV-1A crystal structure showing amino acid residues selected for mutagenesis (yellow sticks) (top panel). The ability of these mutants to inhibit the RNAi response in S2 cells was tested in the RNAi reporter assay (bottom panel). (C) Residues (F114 and P106) in the flexible loop showing strongest inhibition of RNA silencing are highlighted in bold. The RNAi suppression data in (B) and (C) represent mean (±SD) of at least three independent experiments (n = 3) for each condition. (D) The affinity of CrPV-1A proteins for interactions with Ago-2 in S2 cells was probed by Flag-IP and Western blot analysis using antibodies directed against Drosophila Ago-2 protein. Expression levels of CrPV-1A proteins were probed with an anti-Flag antibody. One of three representative experiments is shown (n = 3). (E) S2 cell extracts were incubated with Fluc siRNA, radio-labeled capped Fluc RNA substrate, and purified CrPV-1A or variants. Slicing of the mRNA substrate was detected by denaturing PAGE and autoradiography. One of three representative experiments is shown (n = 3).

Fig 3:. The TALOS element in CrPV-1A shows relaxed specificity for interaction with Ago-2.

(A) List of amino acid insertions and deletions introduced into the CrPV-1A flexible loop. (B) Effects of insertions and deletions on TALOS element-mediated RNAi suppression and Ago-2 interaction were measured using the RNAi reporter assay and Western blot analysis, respectively. (C) Effects of mutation of F114 and P106 for their ability to suppress RNAi and interact with Ago-2 were probed by RNAi-reporter assay and Western blot analysis, respectively. RNAi suppression data in (B) and (C) represent mean (±SD) of at least three independent experiments (n = 3) for each condition. Western blots analysis in (B) and (C) shows one of three representative experiments (n = 3). See also Fig S2

Fig 4:. Importance of the TALOS element in virus pathogenesis.

(A) Schematic representation of the positions (small triangles) at which amino acid mutations were introduced into the CrPV3 infectious cDNA clone. (B) Viral titer by CrPV3 or CrPV3 variant RNA in S2 cells was measured by FFU assay. The synthesis of a viral protein and loading controls were visualized by a Western blot analysis using an antibody raised against CrPV 3CD peptides and tubulin, respectively. (C) Ago-2-depleted S2 cells were transfected with CrPV3, CrPV3-P106A, or CrPV3-F114A RNAs. Viral titer and expression of 3CD protein was measured by FFU assay and Western blot, respectively. Titer value in (B) and (C) represents the mean (±SD) of at three replicate experiments (n = 3). ns, not significant; *p<0.05 (Unpaired t test). The statistical significance represents measurement compared to CrPV3. One of two representative experiments (n = 2) is shown for Western blot analysis. (D) CrPV3 or CrPV3-F114A virus, or PBS were injected into flies (n = 10) of either WT or Ago-2 knockout background. Survival data represents mean of three independent experiments (n = 3). ns, not significant; *p<0.05; ****p<0.0001 (Log-rank test). (E) Viral RNA production in injected flies (panel D) was measured by qPCR. Each data point represents the mean (±SD) of three independent qPCR measurements (n = 3) using infected flies (n = 10). ns, not significant; *p<0.05; ****p<0.0001 (Generalized estimating equation test). See also Fig S3 and Table S2.

Fig 5:. The interactomes of CrPV-1A and Ago-2 protein in S2 cells

(A) For AP-MS experiments, a SaintScore threshold of 0.9, corresponding to a Bayesian False Discovery Rate (BFDR) of < 0.01, was used for reporting specific interactions. Heat map plots of most and least frequently observed interactors for a given bait protein in red and black, respectively. AP-MS experiment represents at least 3 biological replicates per bait protein (n = 3–5) (B) Networks for CrPV-1A, CrPV-1A+Infection, and Ago-2 interacting proteins depicting functionally associated protein clusters were generated using the String database (String Score Threshold = 0.9), and a layout was created using Cytoscape. The yellow dotted circle highlights the most prominent functionally associated protein interactions for CrPV-1A. See also Fig S4 and Table S3.

Fig 6:. CrPV-1A hijacks cellular E3 ligase components.

(A) Glycerol gradient fractions containing CrPV-1A associated proteins were analyzed using SDS-PAGE and silver staining. Gel bands corresponding to Ago-2, Cul2, EloB, EloC, and Rbx1 are indicated with red rectangles, and their identities were verified by mass spectrometry. After fraction 11, HSP27 was observed to run with similar electrophoretic mobility to CrPV-1A as confirmed by mass spectrometry. One of two representative silver stain gels is shown (n = 2). (B) Blue boxes highlight the consensus BC-box amino acid sequences of CrPV-1A, HIV Vif, and BIV Vif. Structural conservation of the BC-box motif for CrPV1A and HIV Vif is shown. The TALOS element residues (P106 and F114) and the BC-box motif are highlighted in yellow and green, respectively. (C) Assembly of CrPV-1A+EloB+EloC ternary and CrPV-1A+EloB+EloC+Cul2 quaternary complexes were examined by overexpression the proteins in E.coli, His-tag pull-down, and Coomassie blue staining. Elution patterns of the complexes was examined by Superdex-G75 and Superdex-200 column chromatography, respectively. See also Fig S5

Fig 7:. CrPV-1A-hijacked E3 ligase contributes to virus replication

(A, B) The stability of Ago-2 in CrPV-1A or CrPV-1A(BC-mut) expressing S2 cells at indicated time points (h) in the presence or absence of MG132 was visualized by Western blot. A lane between 2 and 3 as shown in Fig B has been removed from the original blot for clarity. Western blots analysis are representative of three independent experiments (n = 3) (C) Effects of a single point or combined mutations in the BC-box of the CrPV-1A on RNAi suppression using the RNAi reporter assay. RNAi suppression data represent mean (±SD) of at least three independent experiments (n = 3) for each condition. (D) Red dots represent the ubiquitin sites on Ago-2 derived from S2 cells or S2 cells expressing CrPV-1A protein by UbiScan analysis. The Ubiquitin sites reported are representative of three independent experiments (n = 3). Approximate boundaries of the domains in Drosophila Ago-2 were derived from homology-based modeling using the structure of human Ago-2. (E) S2 cells expressing Ago-2-flag were depleted for Cul2 and EloB followed by infection with CrPV at MOI of 2. Twenty hours post infection, Ago-2 expression in S2 cells and loading controls were analyzed by Western blot using flag and actin antibodies, respectively. Western blots analysis is representative of two independent experiments (n = 2). (F) Cul2-depleted (dsCul2) or Luc-depleted (dsLuc) S2 cells were monitored by infection of CrPV at MOI of 2, and viral titer was measured by FFU assay. Data shows one of three representative experiments with the mean (±SD) of three replicates (n = 3). *p<0.05; ***p<0.001 (Multiple t test). (G) Ago-2-depleted S2 cells were transfected with CrPV3 RNA or CrPV3 variant RNA and viral titer was measured by FFU assay. Each titer value represents the mean (±SD) of at least three replicate experiments (n = 3). The statistical significance represents measurement compared to CrPV3. ns, not significant; *p<0.05 (Unpaired t test) (H) A model showing the mechanism of immune restriction against CrPV (top) and the mechanism deployed by CrPV to counteract this host immune response (bottom) in Drosophila cells. See also Fig S6 and S7, Table S2 and Table S4.