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Resolving the role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in households with members from different generations is crucial for containing the current pandemic. We conducted a large-scale, multicenter, cross-sectional seroepidemiologic household transmission study in southwest Germany during May 11–August 1, 2020. We included 1,625 study participants from 405 households that each had ≥1 child and 1 reverse transcription PCR–confirmed SARS-CoV-2–infected index case-patient. The overall secondary attack rate was 31.6% and was significantly higher in exposed adults (37.5%) than in children (24.6%–29.2%; p = 60 years of age (72.9%; p = 0.039). Other risk factors for infectiousness of the index case-patient were SARS-CoV-2–seropositivity (odds ratio [OR] 27.8, 95% CI 8.26–93.5), fever (OR 1.93, 95% CI 1.14–3.31), and cough (OR 2.07, 95% CI 1.21–3.53). Secondary infections in household contacts generate a substantial disease burden.

Surface residing SARS-CoV-2 is efficiently inactivated by UV-C irradiation. This raises the question whether UV-C-based technologies are also suitable to decontaminate SARS-CoV-2- containing aerosols and which doses are needed to achieve inactivation. Here, we designed a test bench to generate aerosolized SARS-CoV-2 and exposed the aerosols to a defined UV-C dose. Our results demonstrate that the exposure of aerosolized SARS-CoV-2 with a low average dose in the order of 0.42-0.51 mJ/cm

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen's κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.

Purpose To study the possibility of SARS-CoV-2 to infect human corneal cells and tissues under standard corneal culture conditions using explants of COVID-19 donors and primary cornea-derived epithelial cells. Methods Cornea isolated from deceased COVID-19 donors was cultured for 4 weeks, and SARS-CoV-2 replication was monitored by qRT-PCR. Furthermore, primary corneal epithelial cells from healthy donors were cultured ex vivo and infected with SARS-CoV-2 and human cytomegalovirus (HCMV) as a control. Infection status was assessed by western blotting and reporter gene expression using green fluorescent protein–expressing viral strains. ACE2 and TMPRSS2 receptor expression levels in cornea and epithelial cells were assessed by qRT-PCR. Results We did not detect SARS-CoV-2 replication in 10 corneas isolated from deceased COVID-19 patients and cultured for 4 weeks, indicating absence of infection under natural conditions. Furthermore, high-titer SARS-CoV-2 infection of ex vivo cultured cornea-derived epithelial cells did not result in productive virus replication. In contrast, the same cells were highly permissive for HCMV. This phenotype could potentially be explained by low ACE2 and TMPRSS2 transcriptional activity in cornea and cornea-derived epithelial cells. Conclusions Our data suggest that cornea and limbal epithelial cells are refractory to productive SARS-CoV-2 infection. This could be due to the absence of robust receptor expression levels necessary for viral entry. This study adds further evidence to support the very low possibility of transmission of SARS-CoV-2 from an infected corneal transplant donor to a recipient in corneal organ cultures.

BackgroundThe SARS-CoV-2 pandemic urges for cheap, reliable, and rapid technologies for disinfection and decontamination. One frequently proposed method is UV-C irradiation. However, UV-C doses necessary to achieve inactivation of high-titer SARS-CoV-2 are poorly defined.MethodsUsing a box and two handheld systems designed to decontaminate objects and surfaces we evaluated the efficacy of 254 nm UV-C treatment to inactivate surface dried SARS-CoV-2.ResultsDrying for two hours did not have a major impact on the infectivity of SARS-CoV-2, indicating that exhaled virus in droplets or aerosols stays infectious on surfaces at least for a certain amount of time. Short exposure of high titer surface dried virus (3-5*10^6 IU/ml) with UV-C light (16 mJ/cm2) resulted in a total inactivation of SARS-CoV-2. Dose-dependency experiments revealed that 3.5 mJ/cm2 were still effective to achieve a > 6-log reduction in viral titers whereas 1.75 mJ/cm2 lowered infectivity only by one order of magnitude.ConclusionsOur results demonstrate that SARS-CoV-2 is rapidly inactivated by relatively low doses of UV-C irradiation. Furthermore, the data reveal that the relationship between UV-C dose and log-viral titer reduction of surface residing SARS-CoV-2 is non-linear. In the context of UV-C-based technologies used to disinfect surfaces, our findings emphasize the necessity to assure sufficient and complete exposure of all relevant areas by integrated UV-C doses of at least 3.5 mJ/cm2 at 254 nm. Altogether, UV-C treatment is an effective non-chemical possibility to decontaminate surfaces from high-titer infectious SARS-CoV-2.

Even in the face of global vaccination campaigns, there is still an urgent need for effective antivirals against SARS-CoV-2 and its rapidly spreading variants. Several natural compounds show potential as antiviral substances and have the advantages of broad availabilities and large therapeutic windows. Here, we report that lectin from Triticum vulgaris (Wheat Germ Agglutinin) displays antiviral activity against SARS-CoV-2 and its major Variants of Concern (VoC), Alpha and Beta. In Vero B4 cells, WGA potently inhibits SARS-CoV-2 infection with an IC50 of &lt, 10 ng/mL. WGA is effective upon preincubation with the virus or when added during infection. Pull-down assays demonstrate direct binding of WGA to SARS-CoV-2, further strengthening the hypothesis that inhibition of viral entry by neutralizing free virions might be the mode of action behind its antiviral effect. Furthermore, WGA exhibits antiviral activity against human coronavirus OC43, but not against other non-coronaviruses causing respiratory tract infections. Finally, WGA inhibits infection of the lung cell line Calu-3 with wild type and VoC viruses with comparable IC50 values. Altogether, our data indicate that topical administration of WGA might be effective for prophylaxis or treatment of SARS-CoV-2 infections.

Even in the face of global vaccination campaigns, there is still an urgent need for effective antivirals against SARS-CoV-2 and its rapidly spreading variants. Several natural compounds show potential as antiviral substances and have the advantages of broad availabilities and large therapeutic windows. Here, we report that lectin from Triticum vulgaris (Wheat Germ Agglutinin) displays antiviral activity against SARS-CoV-2 and its major Variants of Concern (VoC), Alpha and Beta. In Vero B4 cells, WGA potently inhibits SARS-CoV-2 infection with an IC50 of

The ongoing COVID-19 pandemic and the frequent emergence of new SARS-CoV-2 variants of concern (VOCs), requires continued development of fast and effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nb) specific for the receptor-binding domain (RBD) of SARS-CoV-2, which are now being used as biparatopic Nbs (bipNbs) to investigate their potential as future drug candidates. Following detailedin vitrocharacterization, we chose NM1267 as the most promising candidate showing high affinity binding to several recently described SARS-CoV-2 VOCs and strong neutralizing capacity against a patient isolate of B.1.351 (Beta). To assess if bipNb NM1267 confers protection against SARS-CoV-2 infectionin vivo, human ACE2 transgenic mice were treated by intranasal route before infection with a lethal dose of SARS-CoV-2. NM1267-treated mice showed significantly reduced disease progression, increased survival rates and secreted less infectious virus via their nostrils. Histopathological analyses andin situhybridization further revealed a drastically reduced viral load and inflammatory response in lungs of NM1267-treated mice. These data suggest, that bipNb NM1267 is a broadly active and easily applicable drug candidate against a variety of emerging SARS-CoV-2 VOCs.

Downregulation of ubiquitin (Ub) ligase activity prevents premature ubiquitination and is critical for cellular homeostasis. Nedd4 Ub ligases share a common domain architecture and yet are regulated in distinct ways through interactions of the catalytic HECT domain with the N-terminal C2 domain or the central WW domain region. Smurf1 and Smurf2 are two highly related Nedd4 ligases with ~70% overall sequence identity. Here, we show that the Smurf1 C2 domain interacts with the HECT domain and inhibits ligase activity in trans. However, in contrast to Smurf2, we find that full-length Smurf1 is a highly active Ub ligase, and we can attribute this striking difference in regulation to the lack of one WW domain (WW1) in Smurf1. Using NMR spectroscopy and biochemical assays, we identified the WW1 region as an additional inhibitory element in Smurf2 that cooperates with the C2 domain to enhance HECT domain binding and Smurf2 inhibition. Our work provides important insights into Smurf regulation and highlights that the activities of highly related proteins can be controlled in distinct ways.

In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.

A double punch against SARS-CoV-2 Monoclonal antibodies are an important weapon in the battle against COVID-19. However, these large proteins are difficult to produce in the needed quantities and at low cost. Attention has turned to nanobodies, which are aptly named, single-domain antibodies that are easier to produce and have the potential to be administered by inhalation. Koenig et al. describe four nanobodies that bind to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and prevent infection of cells (see the Perspective by Saelens and Schepens). Structures show that the nanobodies target two distinct epitopes on the SARS-CoV-2 spike protein. Multivalent nanobodies neutralize virus much more potently than single nanobodies, and multivalent nanobodies that bind two epitopes prevent the emergence of viral escape mutants. Science, this issue p. eabe6230; see also p. 681, SARS-CoV-2–neutralizing nanobodies were combined to design potent multivalent nanobodies., INTRODUCTION The global scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose unprecedented challenges to society, health care systems, and science. In addition to effective and safe vaccines, passive immunization by antibody-related molecules offers an opportunity to harness the vertebrate immune system to fight viral infections in high-risk patients. Variable domains of heavy-chain–only antibodies (VHHs), also known as nanobodies, are suitable lead molecules in such efforts, as they are small, extremely stable, easy to engineer, and economic to produce in simple expression systems. RATIONALE We engineered improved multivalent nanobodies neutralizing SARS-CoV-2 on the basis of two principles: (i) detailed structural information of their epitopes and binding modes to the viral spike protein and (ii) mechanistic insights into viral fusion with cellular membranes catalyzed by the spike. RESULTS Nanobodies specific for the receptor binding domain (RBD) of SARS-CoV-2 spike were identified by phage display using nanobody libraries from an alpaca and a llama immunized with the RBD and inactivated virus. Four of the resulting nanobodies—VHHs E, U, V, and W—potently neutralize SARS-CoV-2 and SARS-CoV-2–pseudotyped vesicular stomatitis virus. X-ray crystallography revealed that the nanobodies bind to two distinct epitopes on the RBD, interfaces “E” and “UVW,” which can be synergistically targeted by combinations of nanobodies to inhibit infection. Cryo–electron microscopy (cryo-EM) of trimeric spike in complex with VHH E and VHH V revealed that VHH E stabilizes a conformation of the spike with all three RBDs in the “up” conformation (3-up), a state that is typically associated with activation by receptor binding. In line with this observation, we found that VHH E triggers the fusion activity of spike in the absence of the cognate receptor ACE2. VHH V, by contrast, stabilizes spike in a 2-up conformation and does not induce fusion. On the basis of the structural information, we designed bi- and trivalent nanobodies with improved neutralizing properties. VHH EEE most potently inhibited infection, did not activate fusion, and likely inactivated virions by outcompeting interaction of the virus with its receptor. Yet evolution experiments revealed emergence of escape mutants in the spike with single–amino acid changes that were completely insensitive to inhibition by VHH EEE. VHH VE also neutralized more efficiently than VHH E or VHH V alone; stabilized the 3-up conformation of spike, as determined by cryo-EM; and more strongly induced the spike fusogenic activity. We conclude that the premature activation of the fusion machinery on virions was an unexpected mechanism of neutralization, as enhanced neutralization could not be attributed simply to better blocking of virus-receptor interactions. Activation of spike in the absence of target membranes likely induces irreversible conformational changes to assume the energetically favorable postfusion conformation without catalyzing fusion per se. Simultaneous targeting of two independent epitopes by VHH VE largely prevented the emergence of resistant escape mutants in evolution experiments. CONCLUSION Our results demonstrate the strength of the modular combination of nanobodies for neutralization. Premature activation of spike by nanobodies reveals an unusual mode of neutralization and yields insights into the mechanism of fusion. Bivalent nanobodies neutralize by inducing postfusion conformation of the SARS-CoV-2 spike. On virions, SARS-CoV-2 spike trimers are mostly in an inactive configuration with all RBDs in the down conformation (left). Binding of bivalent nanobody VE stabilizes the spike in an active conformation with all RBDs up (middle), triggering premature induction of the postfusion conformation, which irreversibly inactivates the spike protein (right)., The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Facing the worldwide disease progression of COVID-19 caused by the SARS-CoV-2 virus, the situation is highly critical and there is an unmet need for effective vaccination, reliable diagnosis and therapeutic intervention. Neutralizing binding molecules such as antibodies or derivatives thereof have become important tools for acute treatment of COVID-19. Additionally, such binders provide the unique possibility to monitor the emergence and presence of a neutralizing immune response in infected or vaccinated individuals. Here we describe a set of 11 unique nanobodies (Nbs), originated from an immunized alpaca which bind with high affinities to the glycosylated SARS-CoV-2 Spike receptor domain (RBD). Using a multiplex in vitro binding assay we showed that eight of the selected Nbs effectively block the interaction between RBD, S1-domain and homotrimeric Spike protein with the angiotensin converting enzyme 2 (ACE2) as the viral docking site on human cells. According to competitive binding analysis and detailed epitope mapping, we grouped all Nbs blocking the RBD:ACE2 interaction in three distinct Nb-Sets and demonstrated their neutralizing effect with IC50 values in the low nanomolar range in a cell-based SARS-CoV-2 neutralization assay. Tested Nb combinations from different sets showed substantially lower IC50 values in both functional assays indicating a profound synergistic effect of Nbs simultaneously targeting different epitopes within the RBD. Finally, we applied the most potent Nb combinations in a competitive multiplex binding assay which we termed NeutrobodyPlex and detected a neutralizing immune response in plasma samples of infected individuals. We envisage that our Nbs have a high potential for prophylactic as well as therapeutic options and provide a novel approach to screen for a neutralizing immune response in infected or vaccinated individuals thus helping to monitor the immune status or to guide vaccine design.

SummaryBackgroundPatients infected with SARS-CoV-2 exhibit a highly variable clinical course, varying from barely discernible signs of disease, to moderate flu-like symptoms and, occasionally, with life-threatening pneumonia and/or cytokine storm. The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein as well the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted COVID-19 patients has not been investigated longitudinally so far.MethodsSeveral new serological methods to examine these parameters were developed and validated for the longitudinal investigation in three patients of a family which underwent a mild course of COVID-19.FindingsWe observed that the virus load had almost completely disappeared after about four weeks, whereas serum antibodies showed a contrasting course. IgA levels to S1-RBD-protein and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau approximately six weeks after disease onset. NAbs in serum reached a peak about four weeks after disease onset but dropped to a lower level about six weeks later.InterpretationOur data establishes associations of virus neutralization and a serological immune response foremost against Sars-CoV-2 S1-RDB-protein in a longitudinal manner.

The majority of infections with SARS-CoV-2 (SCoV2) are asymptomatic or mild without the necessity of hospitalization. It is of outmost importance to reveal if these patients develop an antibody response against SCoV2 and to define which antibodies confer virus neutralization. We hence conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against authentic SCoV2 employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SCoV2 and two other sera (4%) were only positive in one of the serological assays employed. For the remainder, antibody response against the S-protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. Only six sera (12%) could be classified as highly neutralizing. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, our data suggest that antibodies against the seasonal coronavirus 229E contribute to SCoV2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SCoV2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SCoV2. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by grants to MS from the Baden-Wuerttemberg foundation (BW-Stiftung), the Deutsche Forschungsgemeinschaft as well as by basic funding provided to MS by the University Hospital Tuebingen and TUFF-Gleichstellungsfoerderung to K.A. (2563-0-0). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: We obtained IRB approval from the local ethics committees #222/2020BO All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All relevant data is within the manuscript and the supplementary information file.

BackgroundSeroreactivity against human endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays that are capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We set up a platform for serum-screening and developed a bead-based Western blot system, namely DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses.MethodsThe parallelized and miniaturised DigiWest assay was adapted for detecting antibodies using whole protein extract prepared from isolated SARS-CoV-2 virus particles. After characterisation and optimization of the newly established test, whole virus lysates of OC43, 229E, and NL63 were integrated into the system.ResultsThe DigiWest-based immunoassay system for detection of SARS-CoV-2 specific antibodies shows a sensitivity of 87.2 % and diagnostic specificity of 100 %. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates a comparable assay performance (Cohen’s Kappa ranging from 0.8799-0.9429). In the multiplexed assay, antibodies against the endemic coronaviruses OC43, 229E, and NL63 were detected, displaying a high incidence of seroreactivity against these coronaviruses.ConclusionThe DigiWest-based immunoassay, which uses authentic antigens from isolated virus particles, is capable of detecting individual serum responses against SARS-CoV-2 with high specificity and sensitivity in one multiplexed assay. It shows high concordance with other commercially available serologic assays. The DigiWest approach enables a concomitant detection of antibodies against different endemic coronaviruses and will help to elucidate the role of these possibly cross-reactive antibodies.

Background: Patients infected with SARS-CoV-2 exhibit a highly variable clinical course, varying from barely discernible signs of disease, to moderate flu-like symptoms and, occasionally, with life-threatening pneumonia and/or cytokine storm. The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein as well the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted Covid-19 patients has not been investigated longitudinally so far. Methods: Several new serological methods to examine these parameters were developed and validated for the longitudinal investigation in three patients of a family which underwent a mild course of COVID-19. Findings: We observed that the virus load had almost completely disappeared after about four weeks, whereas serum antibodies showed a contrasting course. IgA levels to S1-RBD and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau approximately six weeks after disease onset. NAbs in serum reached a peak about four weeks after disease onset but dropped to a lower level about six weeks later. Interpretation: Our data establishes associations of virus neutralization and a serological immune response foremost against Sars-CoV-2 S1-RDB protein in a longitudinal manner. Funding Statement: This study was partly funded by the University Hospital, Tuebingen, and grants from the Baden-Wurttemberg Foundation and the Deutsche Forschungsgemeinschaft (DFG). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: This study was approved by the University of Tubingen.

Human cytomegalovirus (HCMV) infection causes severe illness in newborns and immunocompromised patients. Since treatment options are limited there is an unmet need for new therapeutic approaches. Defensins are cationic peptides, produced by various human tissues, which serve as antimicrobial effectors of the immune system. Furthermore, some defensins are proteolytically cleaved, resulting in the generation of smaller fragments with increased activity. Together, this led us to hypothesize that defensin-derived peptides are natural human inhibitors of virus infection with low toxicity. We screened several human defensin HNP4- and HD5-derived peptides and found HD5(1-9) to be antiviral without toxicity at high concentrations. HD5(1-9) inhibited HCMV cellular attachment and thereby entry and was active against primary as well as a multiresistant HCMV isolate. Moreover, cysteine and arginine residues were identified to mediate the antiviral activity of HD5(1-9). Altogether, defensin-derived peptides, in particular HD5(1-9), qualify as promising candidates for further development as a novel class of HCMV entry inhibitors.AUTHOR SUMMARYDefensins are peptides produced by various human organs which take part in the natural defense against pathogens. Recently, it has been shown that defensins are further cleaved to smaller peptides that have high intrinsic anti-microbial activity. We here challenged the hypothesis that these peptides might have antiviral activity, and due to their presumably natural occurrence, low toxicity. Indeed, we found one peptide fragment that turned out to block the attachment of the human cytomegalovirus (HCMV) to cells. Furthermore, this peptide did not show toxicity in various cellular assays or impede the embryonic development of zebrafish at the concentrations used to block HCMV. This is important, since HCMV is one of the most important viral congenital infections. Altogether, our results hold promise for the development of a new class of antivirals against HCMV.

SummaryNedd4-family ubiquitin ligases are key regulators of cell surface receptor signaling. Their dysregulation is associated with several human diseases, including cancer. Under normal conditions, the activity of various Nedd4 E3s is controlled through an autoinhibitory interaction of the N-terminal C2 domain with the C-terminal catalytic HECT domain. Here, we report the structural and functional framework for this intramolecular interaction. Our nuclear magnetic resonance (NMR) data and biochemical analyses on Smurf2 and Nedd4 show that the C2 domain has the potential to regulate E3 activity by maintaining the HECT domain in a low-activity state where its ability for transthiolation and noncovalent Ub binding are impaired.

Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors., IMPORTANCE The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.

Two-component systems (TCSs) are key players in bacterial signaling, to better understand signal-transmission with molecular detail. The TCS DesK/DesR controls fatty acid desaturation in Bacillus subtilis in response to cold shock and other membrane-altering effectors. We had previously put forward a model of signal-dependent allosteric control of the sensor kinase catalytic activity [1,2]. We have now turned our attention to the response regulator DesR. A canonical activation pathway has been widely accepted to explain phosphorylation-mediated control of response regulator function, allosterically coupling the phosphorylation site to the α4β5α5 surface. However, the structural evidence supporting the main hypotheses is still highly fragmentary. We are now reporting the crystal structure of full-length DesR, in complex with a phosphoryl-mimetic, showing the activated state [3]. Several crystal forms of the receiver domain were determined in the active and inactive configurations, revealing molecular details of the activation switch. Comparative small angle X ray scattering of full-length constructs, structure-guided point mutagenesis, as well as in vitro and in vivo functional analyses, allow us to propose an integral model of DesR activation. The phosphorylation of the receiver domain is allosterically coupled not to one, but two exposed surfaces, independently controlling its dimerization and tetramerization. Notably, a novel surface is shown to be essential for a non-canonical dimerization and activation mechanism. Direct coupling analysis highlights this interface as a shared feature of all NarL/LuxR regulators. This surface is further involved in cognate histidine kinase binding, disclosing a novel view of response regulator allosteric control. With the data we are now reporting, the DesK/DesR signaling pathway becomes, to the best of our knowledge, one of the most thoroughly studied examples of a thermosensor TCS at the molecular and biological levels.

The ongoing COVID-19 pandemic and the emergence of new SARS-CoV-2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nbs) specific for the receptor-binding domain (RBD) of SARS-CoV-2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS-CoV-2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb-treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS-CoV-2 VOCs and represent easily applicable drug candidates.