Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when re-purposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerisation of the RING domain, to switch on the ubiquitination activity of TRIM21 and induce virus neutralisation or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyQ tracts, and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%–50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8–92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity.

Attachment of ubiquitin (Ub) to proteins is one of the most abundant and versatile of all posttranslational modifications and affects outcomes in essentially all physiological processes. RING E3 ligases target E2 Ub-conjugating enzymes to the substrate, resulting in its ubiquitination. However, the mechanism by which a ubiquitin chain is formed on the substrate remains elusive. Here we demonstrate how substrate binding can induce a specific RING topology that enables self-ubiquitination. By analyzing a catalytically trapped structure showing the initiation of TRIM21 RING-anchored ubiquitin chain elongation, and in combination with a kinetic study, we illuminate the chemical mechanism of ubiquitin conjugation. Moreover, biochemical and cellular experiments show that the topology found in the structure can be induced by substrate binding. Our results provide insights into ubiquitin chain formation on a structural, biochemical and cellular level with broad implications for targeted protein degradation.

The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.

HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin-like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS-CoV-2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultrastable scaffold.

No abstract

Background Rapid COVID-19 diagnosis in hospital is essential for patient management and identification of infectious patients to limit the potential for nosocomial transmission. The diagnosis of infection is complicated by 30-50% of COVID-19 hospital admissions with nose/throat swabs testing negative for SARS-CoV-2 nucleic acid, frequently after the first week of illness when SARS-CoV-2 antibody responses become detectable. We assessed the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease in the emergency department. Methods We developed (i) an in vitro neutralization assay using a lentivirus expressing a genome encoding luciferase and pseudotyped with spike (S) protein and (ii) an ELISA test to detect IgG antibodies to nucleocapsid (N) and S proteins from SARS-CoV-2. We tested two lateral flow rapid fingerprick tests with bands for IgG and IgM. We then prospectively recruited participants with suspected moderate to severe COVID-19 and tested for SARS-CoV-2 nucleic acid in a combined nasal/throat swab using the standard laboratory RT-PCR and a validated rapid POC nucleic acid amplification (NAAT) test. Additionally, serum collected at admission was retrospectively tested by in vitro neutralisation, ELISA and the candidate POC antibody tests. We evaluated the performance of the individual and combined rapid POC diagnostic tests against a composite reference standard of neutralisation and standard laboratory based RT-PCR. Results 45 participants had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. Using the composite reference standard, prevalence of COVID-19 disease was 53.3% (24/45). Median age was 73.5 (IQR 54.0-86.5) years in those with COVID-19 disease by our reference standard and 63.0 (IQR 41.0-72.0) years in those without disease. The overall detection rate by rapid NAAT was 79.2% (95CI 57.8-92.9%), decreasing from 100% (95% CI 65.3-98.6%) in days 1-4 to 50.0% (95% CI 11.8-88.2) for days 9-28 post symptom onset. Correct identification of COVID-19 with combined rapid POC diagnostic tests was 100% (95CI 85.8-100%) with a false positive rate of 5.3-14.3%, driven by POC LFA antibody tests. Conclusions Combined POC tests have the potential to transform our management of COVID-19, including inflammatory manifestations later in disease where nucleic acid test results are negative. A rapid combined approach will also aid recruitment into clinical trials and in prescribing therapeutics, particularly where potentially harmful immune modulators (including steroids) are used.