A Requirement for Argonaute 4 in Mammalian Antiviral Defense

Figure 1.. Argonaute 4 (AGO4) Is a Unique and Essential Antiviral Mediator Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1+/+ (black) and Ago1−/− (yellow), (D–F) Ago3+/+ (black) and Ago3−/− (green), or (G–I) Ago4+/+ (black) and Ago4−/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B, or VSV G RNA levels were quantified by qPCR relative to TATA box protein (Tbp). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.

Figure 2.. Antiviral Activity of AGO4 Is Interferon (IFN) Dependent and Independent (A) Ifnb1 in VSV-infected BMDMs as measured by qPCR. (B) IFNβ ELISA of poly(I:C)-treated or virus-infected BMDMs. (C) VSV-induced Isg15 levels in the presence or absence of 10 μg/mL anti-IFNAR (MAR-5A3), as measured by qPCR. (D and E) VSV levels at indicated MOIs for 16 h in the presence or absence of 10 μg/mL anti-IFNAR (MAR-5A3) antibody as quantified by plaque assays (D) or qPCR (E) of VSV-G RNA. (F) Ifnb1 induction in influenza-infected BMDMs as measured by qPCR. (G) Representative flow cytometry plots of influenza-GFP-infected BMDMs (MOI 1, 24 h post-infection [p.i.]). GFP percentages of Annexin V− (non-apoptotic) or Annexin V+ (apoptotic) cells are indicated. (H) Percentage of influenza-GFP+ BMDMs at indicated MOIs for 24 h. (I and J) Influenza Ns1 RNA levels as determined by qPCR (I) or influenza titers (J) as determined by plaque assay. Data are from three combined biological replicates performed in triplicate. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by an unpaired t test or one-way or two-way ANOVA with Bonferroni multiple comparison test. N.S., not significant.

Figure 3.. Ago4 Overexpression Reduces Viral Load (A) Flow cytometry of HEK293T transfected with empty vector (EV) or indicated concentrations of Ago4-Myc for 24 h prior to infection with influenza-GFP at MOI 1 for 24 h. GFP percentages of 7-AAD− (non-apoptotic) cells are indicated. Right: mean and SEM of percentage of Flu-GFP relative to EV control. (B and C) Ago4 and influenza Ns1 RNA expression in HEK293T transfected with EV or 10 μg of Ago4-Myc and infected with Influenza A (MOI 1, 24 h p.i.). (D) Myc immunoblot of Ago1, Ago3, or Ago4 transfected HEK293T cells. (E) Percentage of Flu-GFP+ cells in HEK293T cells transfected with 10 μg of the indicated plasmid and infected at MOI 1 for 24 h. 7-AAD−, relative to EV. (F) Influenza viral titers of mouse embryonic fibroblasts (MEFs) transfected with a negative control (NC) siRNA or vsiRNA against NS1. Data are from three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by an unpaired t test or one-way ANOVA with Bonferroni multiple comparison test.

Figure 4.. Reduction in AGO-Loaded Influenza-Derived RNAs in AGO4-Deficient Macrophages (A) Size distribution and percentage of AGO-bound reads mapped to influenza following infection of BMDMs with influenza or influenza del NS1 (MOI 1, 24h.p.i). (B) Immunoblot of pan-AGO or AGO2 of cell lysates or (C) pan-AGO of immunoprecipitates. (D) Amount of AGO-loaded influenza RNA in influenza-infected BMDMs, relative to WT controls. (E) Quantity of AGO-loaded influenza-derived RNA at indicated nucleotide lengths. (F) Origin of AGO-bound RNA that mapped to the influenza A genome. Genomic segments presented from the 3′ end (left) to the 5′ end (right), and negative (−) or positive (+) strand and scale are indicated. Boxes highlight bona fide vsiRNAs (Li et al., 2016; Qiu et al., 2017). (G) Percentage and size distribution of AGO co-immunoprecipitated influenza RNA in BMDMs infected with influenza delNS1 (MOI 1, 24 h p.i.). (H) Relative influenza HA RNA as quantified by qPCR in MEFs infected with influenza or influenza delNS1 in the presence or absence of 10 μg/mL anti-IFNAR. Data are the mean of two or three biological replicates. Error bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as…

Figure 5.. AGO4 Is an Essential Antiviral Mediator In Vivo (A) Weight loss over time following intranasal infection with influenza A (PR/8/1934, 105 plaque-forming units [pfu]/mouse, n = 10 per group). (B) Hematoxylin and eosin staining of lungs 4 days p.i. Scale bar represents 0.2 μm. (C) Proportion of monocyte and dendritic cell populations infiltrating lungs 4 days p.i., as assessed by flow cytometry. (D) Confocal microscopy of influenza-GFP (green) or wheat germ agglutinin (blue) of lungs 72 h p.i. (105 pfu/mouse). Scale bar represents 350 μm. (E–H) Viral titers of bronchoalveolar lavage fluid (BALF) measured by plaque assay (E), immunoblot of influenza NP protein (F), or influenza PA (G) or NP (H) RNA expression in lungs measured by qPCR 4 days p.i. *p < 0.05, **p < 0.01, and ***p < 0.001, as determined by an unpaired t test.