Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21

Tripartite motif (TRIM) 21 is a cytosolic antibody receptor that neutralizes antibody-coated viruses that penetrate the cell and simultaneously activates innate immunity. Here we show that the conjugation of TRIM21 with K63-linked ubiquitin (Ub-63Ub) catalyzed by the sequential activity of nonredundant E2 Ub enzymes is required for its dual antiviral functions. TRIM21 is first labeled with monoubiquitin (monoUb) by the E2 Ube2W. The monoUb is a substrate for the heterodimeric E2 Ube2N/Ube2V2, resulting in TRIM21-anchored Ub-63Ub. Depletion of either E2 abolishes Ub-63Ub and Ub-48Ub conjugation of TRIM21, NF-κB signaling, and virus neutralization. The formation of TRIM21-Ub-63Ub precedes proteasome recruitment, and we identify an essential role for the 19S-resident and degradation-coupled deubiquitinase Poh1 in TRIM21 neutralization, signaling, and cytokine induction. This study elucidates a complex mechanism of step-wise ubiquitination and deubiquitination activities that allows contemporaneous innate immune signaling and neutralization by TRIM21

Effects of BNT162b2 mRNA vaccine on COVID-19 infection and hospitalisation amongst older people:matched case control study for England

Background: The BNT162b2 mRNA vaccine has been shown to be effective at preventing serious COVID-19 events in clinical trials. There is less evidence on effectiveness in real-world settings, especially for older people. Here, we aimed to estimate vaccine effectiveness in the context of the rapid NHS mass-vaccination programme in England, exploiting age-based vaccination eligibility thresholds to minimise and correct for selection bias. Methods: We studied 170,226 individuals between the ages of 80 and 83 years from community settings outside care homes who received one dose of BNT162b2 mRNA between the 15 and 20 December 2020 and were scheduled a second dose 21 days later. We matched these vaccine recipients to slightly younger (aged 76–79 years) persons not yet eligible to receive the vaccine on gender, area of residence, area deprivation, health status, living arrangements, acute illness, and history of seasonal flu vaccination. We compared their rates of COVID-19 positivity and hospitalisation in the subsequent 45 days. We adjusted for the increasing concentration of COVID-19 positivity in the control population caused by the requirement to have no COVID-19 symptoms prior to vaccination. Results: Emergency hospital admissions were 51.0% (95% confidence interval 19.9 to 69.5%) lower and positive COVID-19 tests were 55.2% (40.8 to 66.8%) lower for vaccinated individuals compared to matched controls 21 to 27 days after first vaccination. Emergency admissions were 75.6% (52.8 to 87.6%) lower, and positive COVID-19 tests were 70.1% (55.1 to 80.1%) lower 35 to 41 days after first vaccination when 79% of participants had received a second dose within 26 days of their first dose. Conclusions: Receipt of the BNT162b2 mRNA vaccine is effective at reducing COVID-19 hospitalisations and infections. The nationwide vaccination of older adults in England with the BNT162b2 mRNA vaccine reduced the burden of COVID-19.</p

Antibody-antigen kinetics constrain intracellular humoral immunity

During infection with non-enveloped viruses, antibodies stimulate immunity from inside cells by activating the cytosolic Fc receptor TRIM21. This intracellular humoral response relies on opsonized viral particles reaching the cytosol intact but the antigenic and kinetic constraints involved are unknown. We have solved the structure of a potent TRIM21-dependent neutralizing antibody in complex with human adenovirus 5 hexon and show how these properties influence immune activity. Structure-guided mutagenesis was used to generate antibodies with 20,000-fold variation in affinity, on-rates that differ by ~50-fold and off-rates by >175-fold. Characterization of these variants during infection revealed that TRIM21-dependent neutralization and NFκB activation was largely unaffected by on-rate kinetics. In contrast, TRIM21 antiviral activity was exquisitely dependent upon off-rate, with sub-μM affinity antibodies nevertheless unable to stimulate signaling because of fast dissociation kinetics. These results define the antibody properties required to elicit an efficient intracellular immune response during viral infection

Corrigendum: Antibody-antigen kinetics constrain intracellular humoral immunity

This Article contains a typographical error in the Methods section under the subheading ‘Crystallization’, where the Protein Data Bank accession code ‘5LDN’ was incorrectly given as ‘5LDV’

TRIM21 Promotes cGAS and RIG-I Sensing of Viral Genomes during Infection by Antibody-Opsonized Virus

<div><p>Encapsidation is a strategy almost universally employed by viruses to protect their genomes from degradation and from innate immune sensors. We show that TRIM21, which targets antibody-opsonized virions for proteasomal destruction, circumvents this protection, enabling the rapid detection and degradation of viral genomes before their replication. TRIM21 triggers an initial wave of cytokine transcription that is antibody, rather than pathogen, driven. This early response is augmented by a second transcriptional program, determined by the nature of the infecting virus. In this second response, TRIM21-induced exposure of the viral genome promotes sensing of DNA and RNA viruses by cGAS and RIG-I. This mechanism allows early detection of an infection event and drives an inflammatory response in mice within hours of viral challenge.</p></div

TRIM21 and antibody promote detection of viral nucleic acids by cytosolic sensors.

<p>(<b>A</b>) 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>B</b>) As (A) except 8 hours. (<b>C</b>) Activation of NFκB by AdV+IgG in the presence of panepoxydone (PPD) or 5<i>Z</i>-7-Oxozeaenol (5Z7O). <b>(D)</b> Induction of cytokine mRNAs 8 hours post-infection with AdV+IgG in the presence or absence of 5Z7O. <b>(E</b> and <b>F)</b> 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. <b>(G)</b> 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). (<b>H</b>) As in (G) except 8 hours. (<b>I</b>) 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). (<b>J</b>) As (I), except 8 hours. (<b>K</b>) cGAS, RIG-I, STING and MAVS mRNA levels in MEF cells 4 hours post challenge with AdV, AdV+IgG, HRV or HRV+IgG. (<b>L</b>) 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). (<b>M</b>) TNFα mRNA levels from samples in (L). (<b>N</b>) TNFα mRNA levels 8 hours post challenge as in (M).</p

TRIM21 mediates multiple waves of immune sensing.

<p>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.</p

Transcriptional profiles induced by different antibody-opsonized viruses are differentially modulated during infection.

<p>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).</p

TRIM21-dependent signaling aids control of viral replication, and promotes inflammation <i>in vivo</i>.

<p>(<b>A</b>) 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>B</b>) Immunoblots for TRIM21 and β-Actin for cells as in (A). (<b>C</b>) 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). (<b>D</b>) As (C) except IFNβ1 mRNA. (<b>E</b>) As (C) except TNFα mRNA. (<b>F</b>) As (C) except IRF7 mRNA. * p < 0.05 and *** p < 0.001.</p

Antibody-opsonized virions have exposed genomes and colocalize with cGAS inside infected cells.

<p>(<b>A</b>) 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>B</b>) As (A) except BrdU-AdV pre-incubated with wt9C12. (<b>C</b>) As (A), except BrdU-AdV pre-incubated with mut9C12. (<b>D</b>) BrdU-positive puncta per cell from confocal images from conditions G-I. *** p < 0.001, n ≥ 100 cells per condition. (<b>E</b>) As (B), except HeLa cells transfected with FLAG-cGAS prior to infection, and also stained with anti-FLAG antibody. Scale bars show 20 μm.</p