Fig 1.
Antibodies potentiate immune sensing during adenovirus (AdV) or rhinovirus (HRV) infection.
(A) Increase in NFκB-driven luciferase activity upon infection of MEF cells with AdV or AdV pre-incubated with human serum IgG. (B) TNFα mRNA levels in MEF cells 8 hours post challenge with AdV. (C) As (A), except using HRV. (D) As (B), except using HRV. (E&F) Induction of TNFα transcription in HRV infected cells treated with rupintrivir (E) or expressing 3C protease (F). (G) NFκB activation in cells challenged with HRV14 or HRV2 in the presence or absence of PEI.
Fig 2.
TRIM21 promotes two waves of innate immune signaling in response to antibody-opsonized virus.
(A) TNFα mRNA levels in MEF cells following challenge with AdV or AdV pre-incubated with human serum IgG (AdV+IgG). (B) TNFα mRNA levels in MEF cells 4 hours post challenge with PBS, IgG, AdV, AdV+IgG, p(I:C). (C) As (B) except 8 hours. (D) TNFα mRNA levels in MEF cells following challenge with HRV or HRV+IgG. (E) TNFα mRNA levels in MEF cells after HRV or HRV+IgG. (F) TNFα mRNA levels in MEF cells challenged with DNA or AdV+IgG at 4 hours and 8 hours. Media either continuously exchanged (Wash, gray striped) or cells incubated as previously (Control, black).
Fig 3.
Transcriptional profiles induced by different antibody-opsonized viruses are differentially modulated during infection.
Transcript levels of immune-related genes at 4 and 8 hours post-infection expressed as a fold change (AdV+IgG relative to AdV alone or HRV+IgG relative to HRV alone). Infection was carried out at an MOI of 20 (AdV) or TCID50 of 125 (HRV).
Fig 4.
Antibody-dependent uncoating reveals viral genomes.
(A) NFκB induction in TRIM21 +/+ and -/- MEFs upon transfection of IgG-coated beads. (B) As (A) except measuring the increase in TNFα mRNA at 4 and 8 hours post-infection in TRIM21 +/+ MEFs. (C) TNFα mRNA in MEF cells 8 hours post challenge with AdV (black squares) or transfected AdV DNA (white triangles). (D) TNFα mRNA levels in MEF cells 4 hours post challenge with IgG, AdV, AdV+IgG, HRV, or HRV+IgG (UT, black bars); or as above but following exposure of AdV and HRV to 500 mJ/cm2 UV irradiation (+UV, gray bars). (E) As (D) except 8 hours. (F) AdV genome copy number at various times post-infection with untreated (UT) or crosslinked (PFA or UV) virus. (G) TNFα mRNA levels in MEF cells treated with AdV+IgG relative to AdV alone (AdV) or AdV crosslinked with PFA pre-incubated with IgG relative to PFA-AdV alone (PFA-AdV). (H) Single-plane confocal micrographs of WT MEF cells 150 min post-infection with EdU-AdV. EdU-labelled vDNA is in green and anti-hexon 9C12 in red. White border delineates cell dimensions imaged by differential interference contrast. (I) Viral DNA puncta within each cell were scored for the presence of hexon labelling. (J) Fields of cells with >2 cells were scored for the presence of viral DNA in the nucleus. ***, P<0.0001 unpaired t-test. (K) AdV GFP copy number in MEF cells following infection with AdV (white circles) or AdV pre-incubated with 9C12 anti-AdV antibody (AdV+wt9C12, black squares). (L) AdV GFP copy number 2 hours post infection with AdV, AdV+wt9C12 or AdV pre-incubated with mutant 9C12 with reduced TRIM21 binding (AdV+mut9C12). (M) As (L), except cells pre-treated with proteasome inhibitor (epoxomicin) or solvent control (DMSO). * p < 0.05. (N) As (L), except PFA-crosslinked AdV.
Fig 5.
Antibody-opsonized virions have exposed genomes and colocalize with cGAS inside infected cells.
(A) Confocal microscope image of MEF cells 2 hours post infection with AdV containing BrdU-labeled DNA (BrdU-AdV), stained with DAPI and anti-BrdU antibody. Scale bars show 20 μm. (B) As (A) except BrdU-AdV pre-incubated with wt9C12. (C) As (A), except BrdU-AdV pre-incubated with mut9C12. (D) BrdU-positive puncta per cell from confocal images from conditions G-I. *** p < 0.001, n ≥ 100 cells per condition. (E) As (B), except HeLa cells transfected with FLAG-cGAS prior to infection, and also stained with anti-FLAG antibody. Scale bars show 20 μm.
Fig 6.
TRIM21 and antibody promote detection of viral nucleic acids by cytosolic sensors.
(A) TNFα mRNA levels 4 hours post challenge of MEF cells with IgG, AdV, AdV pre-incubated with IgG (AdV+IgG), HRV, or HRV pre-incubated with IgG (HRV-IgG); MEF cells treated with negative control scrambled sequence siRNA (NC si, black), or siRNA against MAVS (siMAVS, white) or STING (siSTING, gray). (B) As (A) except 8 hours. (C) Activation of NFκB by AdV+IgG in the presence of panepoxydone (PPD) or 5Z-7-Oxozeaenol (5Z7O). (D) Induction of cytokine mRNAs 8 hours post-infection with AdV+IgG in the presence or absence of 5Z7O. (E and F) Induction of TNFα mRNA at 4hrs (E) or 8hrs (F) post-infection with HRV, HRV+IgG, Adv or Adv+IgG in the presence of PPD, TBK1 inhibitor BX795, or DMSO control. (G) TNFα mRNA levels 4 hours post challenge with IgG, AdV, AdV+IgG, HRV, or HRV+IgG; MEF cells treated with NC si (black), or siRNA directed against RIG-I (si RIG-I, gray checks). (H) As in (G) except 8 hours. (I) TNFα mRNA levels 4 hours post challenge with IgG, AdV or AdV+IgG; MEF cells treated with NC si (black), or siRNA directed against cGAS (si cGAS, gray). (J) As (I), except 8 hours. (K) cGAS, RIG-I, STING and MAVS mRNA levels in MEF cells 4 hours post challenge with AdV, AdV+IgG, HRV or HRV+IgG. (L) cGAS mRNA levels 4 hours post challenge with IgG, AdV, AdV+IgG, HRV or HRV+IgG on MEF cells either without (UT, black) or with simultaneous addition of recombinant IFNα (+IFNα, gray stripes). (M) TNFα mRNA levels from samples in (L). (N) TNFα mRNA levels 8 hours post challenge as in (M).
Fig 7.
TRIM21-dependent signaling aids control of viral replication, and promotes inflammation in vivo.
(A) Viability of HeLa cells or HeLa cells stably depleted of TRIM21 (shT21) following treatment with PPD or 5Z7O then infection with HRV pre-incubated with IgG. (B) Immunoblots for TRIM21 and β-Actin for cells as in (A). (C) IFNα mRNA levels in brain tissue 6 hours post infection, following passive transfer of immune serum (or PBS control) at day -1: PBS control plus mock infection (PBS); immune serum plus mock infection (Serum (S)); PBS control plus MAV-1 infection (MAV-1); immune serum plus MAV-1 infection (MAV-1 +S). (D) As (C) except IFNβ1 mRNA. (E) As (C) except TNFα mRNA. (F) As (C) except IRF7 mRNA. * p < 0.05 and *** p < 0.001.
Fig 8.
TRIM21 mediates multiple waves of immune sensing.
TRIM21 intercepts antibody-bound virions during infection of cells of various hematopoietic or non-hematopoietic lineages, and mediates both innate immune sensing and post-entry neutralization. Upon recognition of intracellular antibody, TRIM21 catalyzes formation of K63-linked polyubiquitin chains, which directly activate components of the NFκB, AP-1 and IRF3/5/7 signaling pathways, resulting in transcription of type I IFN and other innate immune mediators at 4 hours post infection. Concurrent with signaling, TRIM21 recruits p97/VCP and the proteasome to promote premature catastrophic viral uncoating, and degradation of viral proteins. This both prevents productive infection, and exposes viral genomes from within viral capsids to the cytosol. Cytosolic PRRs including cGAS and RIG-I then detect these free viral nucleic acids, activating a second wave of immune transcription at 8 hours post infection.