Lower Serologic Response to COVID-19 mRNA Vaccine in Patients With Inflammatory Bowel Diseases Treated With Anti-TNFα.

BACKGROUND & AIM: Patients with inflammatory bowel diseases (IBD), specifically those treated with anti-tumor necrosis factor (TNF)α biologics, are at high risk for vaccine-preventable infections. Their ability to mount adequate vaccine responses is unclear. The aim of the study was to assess serologic responses to messenger RNA-Coronavirus Disease 2019 vaccine, and safety profile, in patients with IBD stratified according to therapy, compared with healthy controls (HCs). METHODS: Prospective, controlled, multicenter Israeli study. Subjects enrolled received 2 BNT162b2 (Pfizer/BioNTech) doses. Anti-spike antibody levels and functional activity, anti-TNFα levels and adverse events (AEs) were detected longitudinally. RESULTS: Overall, 258 subjects: 185 IBD (67 treated with anti-TNFα, 118 non-anti-TNFα), and 73 HCs. After the first vaccine dose, all HCs were seropositive, whereas ∼7% of patients with IBD, regardless of treatment, remained seronegative. After the second dose, all subjects were seropositive, however anti-spike levels were significantly lower in anti-TNFα treated compared with non-anti-TNFα treated patients, and HCs (both P < .001). Neutralizing and inhibitory functions were both lower in anti-TNFα treated compared with non-anti-TNFα treated patients, and HCs (P < .03; P < .0001, respectively). Anti-TNFα drug levels and vaccine responses did not affect anti-spike levels. Infection rate (∼2%) and AEs were comparable in all groups. IBD activity was unaffected by BNT162b2. CONCLUSIONS: In this prospective study in patients with IBD stratified according to treatment, all patients mounted serologic response to 2 doses of BNT162b2; however, its magnitude was significantly lower in patients treated with anti-TNFα, regardless of administration timing and drug levels. Vaccine was safe. As vaccine serologic response longevity in this group may be limited, vaccine booster dose should be considered.

Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Crystal structure of tomato spotted wilt virus GN reveals a dimer complex formation and evolutionary link to animal-infecting viruses.

Tospoviridae is a family of enveloped RNA plant viruses that infect many field crops, inflicting a heavy global economic burden. These tripartite, single-stranded, negative-sense RNA viruses are transmitted from plant to plant by thrips as the insect vector. The medium (M) segment of the viral genome encodes two envelope glycoproteins, GN and GC, which together form the envelope spikes. GC is considered the virus fusogen, while the accompanying GN protein serves as an attachment protein that binds to a yet unknown receptor, mediating the virus acquisition by the thrips carrier. Here we present the crystal structure of glycoprotein N (GN) from the tomato spotted wilt virus (TSWV), a representative member of the Tospoviridae family. The structure suggests that GN is organized as dimers on TSWV's outer shell. Our structural data also suggest that this dimerization is required for maintaining GN structural integrity. Although the structure of the TSWV GN is different from other bunyavirus GN proteins, they all share similar domain connectivity that resembles glycoproteins from unrelated animal-infecting viruses, suggesting a common ancestor for these accompanying proteins.

A novel de novo heterozygous pathogenic variant in the SDHA gene results in childhood onset bilateral optic atrophy and cognitive impairment.

Isolated defects in the mitochondrial respiratory chain complex II (CII; succinate-ubiquinone oxidoreductase) are extremely rare and mainly result from bi-allelic mutations in one of the nuclear encoded subunits: SDHA, SDHB and SDHD, which comprise CII and the assembly CII factor SDHAF1. We report an adolescent female who presented with global developmental delay, intellectual disability and childhood onset progressive bilateral optic atrophy. Whole exome sequencing of the patient and her unaffected parents identified the novel heterozygous de novo variant c.1984C > T [NM_004168.4] in the SDHA gene. Biochemical assessment of CII in the patient's derived fibroblasts and lymphocytes displayed considerably decreased CII residual activity compared with normal controls, when normalized to the integral mitochondrial enzyme citrate synthase. Protein modeling of the consequent p.Arg662Cys variant [NP-004159.2] suggested that this substitution will compromise the structural integrity of the FAD-binding protein at the C-terminus that will ultimately impair the FAD binding to SDHA, thus decreasing the entire CII activity. Our study emphasizes the role of certain heterozygous SDHA mutations in a distinct clinical phenotype dominated by optic atrophy and neurological impairment. This is the second mutation that has been reported to cause this phenotype. Furthermore, it adds developmental delay and cognitive disability to the expanding spectrum of the disorder. We propose to add SDHA to next generation sequencing gene panels of optic atrophy.

Pseudoviruses for the assessment of coronavirus disinfection by ozone.

The COVID-19 pandemic has severely impacted public health worldwide. Evidence of SARS-CoV-2 transmission via aerosols and surfaces has highlighted the need for efficient indoor disinfection methods. For instance, the use of ozone gas as a safe and potent disinfectant against SARS-CoV-2 virus is of particular interest. Here we tested the use of pseudoviruses as a model for evaluating ozone disinfection of the coronavirus at ozone concentrations of 30, 100, and 1000 ppmv. Results show that ozone disinfection rate of pseudoviruses was similar to that of coronavirus 229E (HuCoV-229E) at short contact times, below 30 min. Viral infection decreased by 95% following ozone exposure for 20 min at 1000 ppmv, 30 min at 100 ppmv and about 40 min at 30 ppmv. This findings mean that ozone is a powerful disinfectant toward the enveloped pseudovirus even at low ozone exposure. We also showed that viral disinfection occurs on various contaminated surfaces, with a positive association between disinfection and surface hydrophilicity. Infected surfaces made of aluminum alloy, for example, were better disinfected with ozone as compared to brass, copper, and nickel surfaces. Lastly, we demonstrate the advantage of ozone over liquid disinfectants by showing similar viral disinfection on top, side, bottom, and interior surfaces. Overall, our study demonstrates the potential use of ozone gas disinfection to combat the COVID-19 outbreak.

Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation.

Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens.

Crystal structure of glycoprotein C from Rift Valley fever virus.

Rift Valley fever virus (RVFV), like many other Bunyaviridae family members, is an emerging human and animal pathogen. Bunyaviruses have an outer lipid envelope bearing two glycoproteins, G(N) and G(C), required for cell entry. Bunyaviruses deliver their genome into the host-cell cytoplasm by fusing their envelope with an endosomal membrane. The molecular mechanism of this key entry step is unknown. The crystal structure of RVFV G(C) reveals a class II fusion protein architecture found previously in flaviviruses and alphaviruses. The structure identifies G(C) as the effector of membrane fusion and provides a direct view of the membrane anchor that initiates fusion. A structure of nonglycosylated G(C) reveals an extended conformation that may represent a fusion intermediate. Unanticipated similarities between G(C) and flavivirus envelope proteins reveal an evolutionary link between the two virus families and provide insights into the organization of G(C) in the outer shell of RVFV.

Selective pressure causes an RNA virus to trade reproductive fitness for increased structural and thermal stability of a viral enzyme.

The modulation of fitness by single mutational substitutions during environmental change is the most fundamental consequence of natural selection. The antagonistic tradeoffs of pleiotropic mutations that can be selected under changing environments therefore lie at the foundation of evolutionary biology. However, the molecular basis of fitness tradeoffs is rarely determined in terms of how these pleiotropic mutations affect protein structure. Here we use an interdisciplinary approach to study how antagonistic pleiotropy and protein function dictate a fitness tradeoff. We challenged populations of an RNA virus, bacteriophage Φ6, to evolve in a novel temperature environment where heat shock imposed extreme virus mortality. A single amino acid substitution in the viral lysin protein P5 (V207F) favored improved stability, and hence survival of challenged viruses, despite a concomitant tradeoff that decreased viral reproduction. This mutation increased the thermostability of P5. Crystal structures of wild-type, mutant, and ligand-bound P5 reveal the molecular basis of this thermostabilization--the Phe207 side chain fills a hydrophobic cavity that is unoccupied in the wild-type--and identify P5 as a lytic transglycosylase. The mutation did not reduce the enzymatic activity of P5, suggesting that the reproduction tradeoff stems from other factors such as inefficient capsid assembly or disassembly. Our study demonstrates how combining experimental evolution, biochemistry, and structural biology can identify the mechanisms that drive the antagonistic pleiotropic phenotypes of an individual point mutation in the classic evolutionary tug-of-war between survival and reproduction.

The organization of a CSN5-containing subcomplex of the COP9 signalosome.

The COP9 signalosome (CSN) is an evolutionarily conserved multi-protein complex that interfaces with the ubiquitin-proteasome pathway and plays critical developmental roles in both animals and plants. Although some subunits are present only in an ∼320-kDa complex-dependent form, other subunits are also detected in configurations distinct from the 8-subunit holocomplex. To date, the only known biochemical activity intrinsic to the complex, deneddylation of the Cullin subunits from Cullin-RING ubiquitin ligases, is assigned to CSN5. As an essential step to understanding the structure and assembly of a CSN5-containing subcomplex of the CSN, we reconstituted a CSN4-5-6-7 subcomplex. The core of the subcomplex is based on a stable heterotrimeric association of CSN7, CSN4, and CSN6, requiring coexpression in a bacterial reconstitution system. To this heterotrimer, we could then add CSN5 in vitro to reconstitute a quaternary complex. Using biochemical and biophysical methods, we identified pairwise and combinatorial interactions necessary for the formation of the CSN4-5-6-7 subcomplex. The subcomplex is stabilized by three types of interactions: MPN-MPN between CSN5 and CSN6, PCI-PCI between CSN4 and CSN7, and interactions mediated through the CSN6 C terminus with CSN4 and CSN7. CSN8 was also found to interact with the CSN4-6-7 core. These data provide a strong framework for further investigation of the organization and assembly of this pivotal regulatory complex.

RNA Encapsulation Mode and Evolutionary Insights from the Crystal Structure of Emaravirus Nucleoprotein.

Enveloped RNA viruses are rare among plant viruses. Fimoviridae is a newly founded family of plant viruses within the Bunyavirales order that inflicts diverse crop losses worldwide. The fig mosaic virus (FMV), the representative member of the Fimoviridae family, was shown to be a causative agent for the fig mosaic disease. Like all bunyaviruses, FMV has a segmented, negative-sense, single-stranded RNA (ssRNA) genome that is encapsulated by the viral nucleoprotein (N). Here, we present high-resolution crystal structures of FMV N in its RNA-free and RNA-bound forms, revealing a "paper fortune teller" structural transition between the two states. The tightly packed tetramer of FNV N is similar to the structures of other N proteins of different members of the Bunyavirales order. In its RNA-bound form, the tetramer reorganizes to adopt a more open state that allows the accommodation of the RNA. Despite the low sequence similarity to N proteins of animal-infecting bunyaviruses, there is a striking structural resemblance between FMV N and nucleoproteins from members of the Peribunyaviridae, an animal-infecting family of viruses. This structural homology implies that enveloped plant viruses and animal-infecting viruses might have a common ancestor from which they diverged. IMPORTANCE Most insect-born viruses circulate within the Animalia kingdom, whereas plant-infecting RNA viruses are cross-kingdom pathogens. Many plant-infecting viruses cause devastating crop damage that leads to food security endangerment. The evolutionary crossroads of interkingdom circulation and infection are poorly understood. Thus, we took the structural approach to understand the similarities and differences between interkingdom-infecting viruses and viruses that circulate within one kingdom of life. Using our structures of FMV N in its free form and in complex with a single-stranded RNA (ssRNA), we dissected the mechanism by which FMV N binds to the RNA and revealed the conformational changes associated with the binding. The resemblance of our structure to N proteins from members of the Peribunyaviridae family and their recently published ribonucleoprotein (RNP) pseudoatomic resolution assembly model suggests that the FMV genome is similarly encapsulated. Thus, our finding unveils yet another bridge by which plant- and animal-infecting viruses are interconnected.

Developing a peptide to disrupt cohesin head domain interactions.

Cohesin mediates the 3-D structure of chromatin and is involved in maintaining genome stability and function. The cohesin core comprises Smc1 and Smc3, elongated-shaped proteins that dimerize through globular domains at their edges, called head and hinge. ATP binding to the Smc heads induces their dimerization and the formation of two active sites, while ATP hydrolysis results in head disengagement. This ATPase cycle is essential for driving cohesin activity. We report on the development of the first cohesin-inhibiting peptide (CIP). The CIP binds Smc3 in vitro and inhibits the ATPase activity of the holocomplex. Treating yeast cells with the CIP prevents cohesin's tethering activity and, interestingly, leads to the accumulation of cohesin on chromatin. CIP3 also affects cohesin activity in human cells. Altogether, we demonstrate the power of peptides to inhibit cohesin in cells and discuss the potential application of CIPs as a therapeutic approach.

Rapid electrochemical immunodetection of SARS-CoV-2 using a pseudo-typed vesicular stomatitis virus model.

The COVID-19 pandemic has highlighted the need for reliable and accurate diagnostic tools that provide quantitative results at the point of care. Real-time RT-PCR requires large laboratories, a skilled workforce, complex and costly equipment, and labor-intensive sample processing. Despite tremendous efforts, scaling up RT-PCR tests is seemingly unattainable. To date, hundreds of millions of COVID-19 tests have been performed globally, but the demand for timely, accurate testing continues to outstrip supply. Antigen-based rapid diagnostic testing is emerging as an alternative to RT-PCR. However, the performance of these tests, namely their sensitivity, is still inadequate. To overcome the limitations of currently employed diagnostic tests, new tools that are both sensitive and scalable are urgently needed. We have developed a miniaturized electrochemical biosensor based on the integration of specific monoclonal antibodies with a biochip and a measurement platform, and applied it in the detection of Spike S1 protein, the binding protein of SARS-CoV-2. Using electrochemical impedance spectroscopy, quantitative detection of sub-nanomolar concentrations of Spike S1 was demonstrated, exhibiting a broad detection range. To demonstrate the applicability of the biosensor, we have further developed a SARS-CoV-2 pseudovirus based on Spike protein-pseudo-typed VSV platform. Specific detection of different concentrations of pseudovirus particles was feasible in <30 min. This new tool may largely contribute to the fight against COVID-19 by enabling intensive testing to be performed and alleviating most of the hurdles that plague current diagnostics.

A Novel Homozygous Missense Variant in the LRRC32 Gene Is Associated With a New Syndrome of Cleft Palate, Progressive Vitreoretinopathy, Growth Retardation, and Developmental Delay.

Cleft lip and/or cleft palate are a common group of birth defects that further classify into syndromic and non-syndromic forms. The syndromic forms are usually accompanied by additional physical or cognitive abnormalities. Isolated cleft palate syndromes are less common; however, they are associated with a variety of congenital malformations and generally have an underlying genetic etiology. A single report in 2019 described a novel syndrome in three individuals, characterized by cleft palate, developmental delay and proliferative retinopathy due to a homozygous non-sense mutation in the LRRC32 gene encoding glycoprotein A repetitions predominant (GARP), a cell surface polypeptide crucial for the processing and maturation of transforming growth factor ß (TGF-ß). We describe a patient who presented with cleft palate, prenatal and postnatal severe growth retardation, global developmental delay, dysmorphic facial features and progressive vitreoretinopathy. Whole exome sequencing (WES) revealed a very rare homozygous missense variant in the LRRC32 gene, which resulted in substitution of a highly conserved isoleucine to threonine. Protein modeling suggested this variant may negatively affect GARP function on latent TGF-ß activation. In summary, our report further expands the clinical features of cleft palate, proliferative retinopathy and developmental delay syndrome and emphasizes the association of LRRC32 pathogenic variants with this new syndrome.

Conformational flexibility in neutralization of SARS-CoV-2 by naturally elicited anti-SARS-CoV-2 antibodies.

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.

HCV Infection Increases the Expression of ACE2 Receptor, Leading to Enhanced Entry of Both HCV and SARS-CoV-2 into Hepatocytes and a Coinfection State.

Recent studies suggest the enhancement of liver injury in COVID-19 patients infected with Hepatitis C virus (HCV). Hepatocytes express low levels of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor, raising the possibility of HCV-SARS-CoV-2 coinfection in the liver. This work aimed to explore whether HCV and SARS-CoV-2 coinfect hepatocytes and the interplay between these viruses. We demonstrate that SARS-CoV-2 coinfects HCV-infected Huh7.5 (Huh7.5HCV) cells. Both viruses replicated efficiently in the coinfected cells, with HCV replication enhanced in coinfected compared to HCV-mono-infected cells. Strikingly, Huh7.5HCV cells were eight fold more susceptible to SARS-CoV-2 pseudoviruses than naive Huh7.5 cells, suggesting enhanced SARS-CoV-2 entry into HCV-preinfected hepatocytes. In addition, we observed increased binding of spike receptor-binding domain (RBD) protein to Huh7.5HCV cells, as well as enhanced cell-to-cell fusion of Huh7.5HCV cells with spike-expressing Huh7.5 cells. We explored the mechanism of enhanced SARS-CoV-2 entry and identified an increased ACE2 mRNA and protein levels in Huh7.5HCV cells, primary hepatocytes, and in data from infected liver biopsies obtained from database. Importantly, higher expression of ACE2 increased HCV infection by enhancing its binding to the host cell, underscoring its role in the HCV life cycle as well. Transcriptome analysis revealed that shared host signaling pathways were induced in HCV-SARS-CoV-2 coinfection. This study revealed complex interactions between HCV and SARS-CoV-2 infections in hepatocytes, which may lead to the increased liver damage recently reported in HCV-positive COVID-19 patients. IMPORTANCE Here, we provide the first experimental evidence for the coexistence of SARS-CoV-2 infection with HCV, and the interplay between them. The study revealed a complex relationship of enhancement between the two viruses, where HCV infection increased the expression of the SARS-CoV-2 entry receptor ACE2, thus facilitating SARS-CoV-2 entry, and potentially, also HCV entry. Thereafter, SARS-CoV-2 infection enhanced HCV replication in hepatocytes. This study may explain the aggravation of liver damage that was recently reported in COVID-19 patients with HCV coinfection and suggests preinfection with HCV as a risk factor for severe COVID-19. Moreover, it highlights the possible importance of HCV treatment for coinfected patients. In a broader view, these findings emphasize the importance of identifying coinfecting pathogens that increase the risk of SARS-CoV-2 infection and that may accelerate COVID-19-related co-morbidities.

Anti-TNFα Treatment Impairs Long-Term Immune Responses to COVID-19 mRNA Vaccine in Patients with Inflammatory Bowel Diseases.

Patients with inflammatory bowel disease (IBD) treated with anti-tumor-necrosis factor-alpha (TNFα) exhibited lower serologic responses one-month following the second dose of the COVID-19 BNT162b2 vaccine compared to those not treated with anti-TNFα (non-anti-TNFα) or to healthy controls (HCs). We comprehensively analyzed long-term humoral responses, including anti-spike (S) antibodies, serum inhibition, neutralization, cross-reactivity and circulating B cell six months post BNT162b2, in patients with IBD stratified by therapy compared to HCs. Subjects enrolled in a prospective, controlled, multi-center Israeli study received two BNT162b2 doses. Anti-S levels, functional activity, specific B cells, antigen cross-reactivity, anti-nucleocapsid levels, adverse events and IBD disease score were detected longitudinally. In total, 240 subjects, 151 with IBD (94 not treated with anti-TNFα and 57 treated with anti-TNFα) and 89 HCs participated. Six months after vaccination, patients with IBD treated with anti-TNFα had significantly impaired BNT162b2 responses, specifically, more seronegativity, decreased specific circulating B cells and cross-reactivity compared to patients untreated with anti-TNFα. Importantly, all seronegative subjects were patients with IBD; of those, >90% were treated with anti-TNFα. Finally, IBD activity was unaffected by BNT162b2. Altogether these data support the earlier booster dose administration in these patients.

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

Structural basis for UFM1 transfer from UBA5 to UFC1.

Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1's active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1's conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

BNT162b2 mRNA vaccine elicited antibody response in blood and milk of breastfeeding women.

The importance of breastmilk in postnatal life lies in the strong association between breastfeeding and the reduction in the risk of infection and infection-related infant mortality. However, data regarding the induction and dynamics of breastmilk antibodies following administration of the Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine is scarce, as pregnant and lactating women were not included in the initial vaccine clinical trials. Here, we investigate the dynamics of the vaccine-specific antibody response in breastmilk and serum in a prospective cohort of ten lactating women who received two doses of the mRNA vaccine. We show that the antibody response is rapid and highly synchronized between breastmilk and serum, reaching stabilization 14 days after the second dose. The response in breastmilk includes both IgG and IgA with neutralization capacity.

Crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion.

Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the "pH sensor" that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.