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Antibodies predating infection Immunological memory after infection with seasonal human coronaviruses (hCoVs) may potentially contribute to cross-protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Ng et al. report that in a cohort of 350 SARS-CoV-2–uninfected individuals, a small proportion had circulating immunoglobulin G (IgG) antibodies that could cross-react with the S2 subunit of the SARS-CoV-2 spike protein (see the Perspective by Guthmiller and Wilson). By contrast, COVID-19 patients generated IgA, IgG, and IgM antibodies that recognized both the S1 and S2 subunits. The anti-S2 antibodies from SARS-CoV-2–uninfected patients showed specific neutralizing activity against both SARS-CoV-2 and SARS-CoV-2 S pseudotypes. A much higher percentage of SARS-CoV-2–uninfected children and adolescents were positive for these antibodies compared with adults. This pattern may be due to the fact that children and adolescents generally have higher hCoV infection rates and a more diverse antibody repertoire, which may explain the age distribution of COVID-19 susceptibility. Science, this issue p. 1339; see also p. 1272, SARS-CoV-2 neutralizing antibodies can be found in some uninfected individuals—predominantly children and adolescents., Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)–reactive antibodies were detectable using a flow cytometry–based method in SARS-CoV-2–uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S–reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2–uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Several common-cold coronaviruses (HCoVs) are endemic in humans and several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current Coronavirus disease 2019 (COVID-19) pandemic. Whilst antibody cross-reactivity with the Spike glycoproteins (S) of diverse coronaviruses has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to or mediate protection, when induced naturally or through vaccination. Using a mouse model, we show that prior HCoV-OC43 S immunity primes neutralising antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, mouse vaccination with SARS-CoV-2 S2 elicits antibodies that neutralise diverse animal and human alphacoronaviruses and betacoronaviruses in vitro, and protects against SARS-CoV-2 challenge in vivo. Lastly, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induces stronger and broader neutralising antibody response than booster Wuhan S vaccination, suggesting it may prevent repertoire focusing caused by repeated homologous vaccination. The data presented here establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern (VOCs), as well as to unpredictable, yet inevitable future coronavirus zoonoses.

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Background:The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood.Methods:We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity.Results:Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection.Conclusions:The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric.Funding:This work was supported by the Francis Crick Institute and the Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg.

SummaryThe coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterised pharmaceuticals for nsp13 inhibitors using a FRET-based high-throughput screening (HTS) approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

We examined the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 that arose in the United Kingdom and spread globally. Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa B.1.351 variant, than of the infecting variant. The drop in cross-reactivity was more pronounced following B.1.1.7 than parental strain infection, indicating asymmetric heterotypic immunity induced by SARS-CoV-2 variants.

SARS-CoV-2 spike N-terminal domain harbors a potent epitope that can be modulated by binding of natural linear tetrapyrroles., The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo–electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. LC3 is attached to membranes by covalent lipidation that depends on recruitment of the ATG5-12-16L1 complex. Non-canonical LC3 lipidation requires the C-terminal WD40 domain of ATG16L1 that is dispensable for canonical autophagy. We devised a subtractive CRISPR knock-out screening strategy to investigate the requirements for non-canonical LC3-lipidation. This correctly identified the enzyme complexes directly responsible for LC3-lipidation. We additionally identified the RALGAP complex as important for M2-induced, but not ionophore drug induced LC3 lipidation. In contrast, we identified ATG4D as responsible for LC3 recycling in M2-induced and basal LC3-lipidation. Identification of a vacuolar ATPase subunit in the screen suggested a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug induced LC3-lipidation leads to association of the vacuolar ATPase and ATG16L1 and can be antagonised by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.

Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections1. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions2,3. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Summary Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens., Graphical abstract, Highlights • Subtractive CRISPR screen identifies genes involved in non-canonical LC3 lipidation • v-ATPase regulates LC3 lipidation at erroneously neutral compartments • RALGAP complex involved in M2 proton channel induced LC3 lipidation • ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation, Ulferts et al. identify v-ATPase as the central regulator of ATG16L1 WD40 domain-dependent of LC3 lipidation. This lipidation can be prevented by Salmonella SopF and counteracted by ATG4D, the predominant ATG4 paralog responsible for LC3 delipidation. The RalGAP complex affects influenza virus M2-induced LC3 lipidation by affecting M2 trafficking.

Somatic hypermutation of immunoglobulin genes occurs at both C.G pairs and A.T pairs. Mutations at C.G pairs are created by activation-induced deaminase (AID)-catalysed deamination of C residues to U residues. Mutations at A.T pairs are probably produced during patch repair of the AID-generated U.G lesion, but they occur through an unknown mechanism. Here, we compare the popular suggestion of nucleotide mispairing through polymerase error with an alternative possibility, mutation through incorporation of dUTP (or another non-canonical nucleotide).

Human APOBEC3F and APOBEC3G are double-domained deaminases that can catalyze dC--dU deamination in HIV-1 and MLV retroviral DNA replication intermediates, targeting T-C or C-C dinucleotides, respectively. HIV-1 antagonizes their action through its vif gene product, which has been shown (at least in the case of APOBEC3G) to interact with the N-terminal domain of the deaminase, triggering its degradation. Here, we compare APOBEC3F and APOBEC3G to APOBEC3C, a single-domained deaminase that can also act on both HIV-1 and MLV. We find that whereas APOBEC3C contains all the information necessary for both Vif-binding and cytidine deaminase activity in a single domain, it is the C-terminal domain of APOBEC3F and APOBEC3G that confer their target site specificity for cytidine deamination. We have exploited the fact that APOBEC3C, whilst highly homologous to the C-terminal domain of APOBEC3F, exhibits a distinct target site specificity (preferring Y-C dinucleotides) in order to identify residues in APOBEC3F that might affect its target site specificity. We find that this specificity can be altered by single amino acid substitutions at several distinct positions, suggesting that the strong dependence of APOBEC3-mediated deoxycytidine deamination on the 5'-flanking nucleotide is sensitive to relatively subtle changes in the APOBEC3 structure. The approach has allowed the isolation of APOBEC3 DNA mutators that exhibit novel target site preferences.

Background In autophagy, cellular components are engulfed in a double membrane and recycled for their nutritional value. This mechanism has been adapted to defend cells against intracellular pathogens. Although viruses must evade autophagocytic destruction, they can also benefit from the resources that autophagy provides. We explored the molecular mechanisms by which influenza manipulates the autophagocytic machinery. Methods We used pull-down and fluorescence anisotropy assays to investigate the binding of the influenza A virus M2 proton channel to the essential autophagy protein LC3. The distribution of autophagosomes in infected cells was investigated by confocal microscopy. Construction of LIR-mutant viruses allowed us to investigate the importance of the interaction for subverting autophagy and the consequences for virion morphology and stability. Findings We found that binding to LC3 is mediated by a highly conserved LC3 interacting region (LIR) in the cytoplasmic tail of M2, the first such motif discovered in a pathogen. The M2 LIR is required for the redistribution of LC3 to the unusual destination of the plasma membrane in virus-infected cells. Mutations in M2 that abolish binding to LC3 interfere with filamentous budding and reduce virion stability. Interpretation We conclude that influenza A virus subverts autophagy by mimicking a host short linear protein–protein interaction motif. We suggest that this process occurs to provide adequate supply of membrane for virion production. This efficient strategy might facilitate transmission of infection between organisms by enhancing the stability of viral progeny. Funding Academy of Medical Sciences.

An antiviral Dicer defends stem cells Stem cells have a pivotal role in maintaining tissue architecture, integrity, and renewal. Poirier et al. demonstrate that mammalian stem cells can protect themselves from some RNA viruses by expressing an alternatively spliced isoform of the enzyme Dicer called aviD, which potentiates antiviral RNA interference (see the Perspective by Shahrudin and Ding). aviD acts by cleaving long, base-paired viral RNAs to generate small interfering RNAs that direct the sequence-specific cleavage of homologous viral RNAs. This process is reminiscent of that in insects and worms, which also use Dicer-dependent RNA interference in antiviral defense, and contrasts with mammalian differentiated cells, which generally rely on the interferon system to combat virus infection. Science , abg2264, this issue p. 231 ; see also abj5673, p. 160