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Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme. We determined the cryo-electron microscopy structures of Dot1L-H2BK34ub nucleosome complex and the H2BK34ub nucleosome alone. The structures reveal that H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on nucleosome as H2BK120ub, which positions Dot1L for the productive conformation through direct ubiquitin-enzyme contacts. However, H2BK34-anchored ubiquitin does not directly interact with Dot1L as occurs in the case of H2BK120ub, but rather induces DNA and histone distortion around the modified site. Our findings establish the structural framework for understanding the H2BK34ub-H3K79me trans-crosstalk and highlight the diversity of mechanisms for histone ubiquitination to activate chromatin-modifying enzymes.

The persistence of the coronavirus disease 2019 (COVID-19) pandemic has resulted in increasingly disruptive impacts, and it has become the most devastating challenge to global health in a century. The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants challenges the currently available therapeutics for clinical application. Nonstructural proteins (also known as replicase proteins) with versatile biological functions play central roles in viral replication and transcription inside the host cells, and they are the most conserved target proteins among the SARS-CoV-2 variants. Specifically, they constitute the replication–transcription complexes (RTCs) dominating the synthesis of viral RNA. Knowledge of themolecular mechanisms of nonstructural proteins and their assembly into RTCs will benefit the development of antivirals targeting them against existing or potentially emerging variants. In this review, we summarize current knowledge of the structures and functions of coronavirus nonstructural proteins as well as the assembly and functions of RTCs in the life cycle of the virus.

Nonsmall cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases (more than 1.85 million cases with 1.48 million deaths in 2020). In the present study, two novel oncolytic SVV mutants modified based on structural biology and reverse genetics (viral receptor-associated mutant SVV-S177A and viral antigenic peptide-related mutant SVV-S177A/P60S) with increased infectivity or lower immunogenicity significantly ( P < 0.001) prolonged the mOS from 11 days in the control cohort to 23 days in the SVV-S177A cohort and the SVV-S177A/P60S cohort in the NSCLC-bearing athymic mouse model, which may provide the direction for modifying SVV to improve the effect of oncolysis.

COVID-19 altered our lives and pushed scientific research to operate at breakneck speed, leading to significant breakthroughs in record time. We asked experts in the field about the challenges they faced in transitioning, rapidly but safely, to working on the virus while navigating the shutdown. Their voices converge on the importance of teamwork, forging new collaborations, and working toward a shared goal.

Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5′-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription., SARS-CoV-2 virus replication and transcription is mediated by the replication and transcription complex (RTC) that is composed of 16 non-structural proteins (nsp). Here, the authors present the cryo-EM structure of a SARS-CoV-2 mini RTC consisting of the viral RNA-dependent RNA polymerase with a template-primer RNA, the RdRp cofactors nsp7 and nsp8 and two nsp13 helicase molecules, and they propose a model for helicase-polymerase coupling during SARS-CoV-2 RTC assembly.

Segmented negative-sense RNA viruses (sNSRVs) encode a single-polypeptide polymerase (L protein) or a heterotrimeric polymerase complex to cannibalize host messenger RNA cap structures serving as primers of transcription, and catalyse RNA synthesis. Here, we report the full-length structure of the severe fever with thrombocytopaenia syndrome virus (SFTSV) L protein, as determined by cryogenic electron microscopy at 3.4 A, leading to an atomic model harbouring three functional parts (an endonuclease, an RNA-dependent RNA polymerase and a cap-binding domain) and two structural domains (an arm domain with a blocker motif and a carboxy-terminal lariat domain). The SFTSV L protein has a compact architecture in which its cap-binding pocket is surprisingly occupied by an Arg finger of the blocker motif, and the endonuclease active centre faces back towards the cap-binding pocket, suggesting that domain rearrangements are necessary to acquire the pre-initiation state of the active site. Our results provide insight into the complete architecture of sNSRV-encoded L protein and further the understanding of sNSRV transcription initiation. The authors present the full-length structure of the polymerase (L protein) from the severe fever with thrombocytopenia syndrome virus (SFTSV)—a segmented, negative-stranded RNA virus. The structure reveals important details with implications for the understanding of viral transcription and replication, and provides information on the organization of the polymerase core domain, entrance and exit tunnels, as well as the cap-binding and endonuclease domains.

Upon sensing pathogen-associated patterns and secreting interferons (IFNs) into the environment, host cells perceive extracellular type I IFNs by the IFNα/β receptors IFNAR1 and IFNAR2 to stimulate downstream innate immune signaling cascades. Through the use of chemical probes, we demonstrated that IFNAR2 facilitates hepatitis C virus (HCV) entry. Silencing of IFNAR2 significantly attenuated HCV proliferation. IFNAR2 binds infectious HCV virions through a direct interaction of its D2 domain with the C-terminal end of apolipoprotein E (apoE) on the viral envelope and facilitates virus entry into host cells. The antibody against the IFNAR2 D2 domain attenuates IFNAR2-apoE interaction and impairs HCV infection. The recombinant IFNAR2 protein and the chemical probe potently inhibit major HCV genotypes in various human liver cells in vitro. Moreover, the impact of a chemical probe on HCV genotype 2a is also documented in immune-compromised humanized transgenic mice. Our results not only expand the understanding of the biology of HCV entry and the virus-host relationship but also reveal a new target for the development of anti-HCV entry inhibitors.

Abscisic acid (ABA) is a key phytohormone with diverse functions in plants, and its signal transduction is mainly mediated by ABA receptors termed PYR/PYL/RCARs (hereafter referred to as PYLs) through the PYLs-PP2Cs-SnRK2s regulatory systems. However, the model failed to account for the roles of some important known regulators of ABA physiology. Given the central role of PYLs in ABA signal transduction, we therefore speculated that ABA receptors PYLs might be involved in regulatory pathways other than PP2Cs. Thus, a comprehensive analysis of PYLs-interacting partners could greatly facilitate the identification of unknown regulatory pathways, advancing our knowledge of the ABA signaling mechanism. Herein, we present a strategy involving covalent chemical capture coupled with HPLC-MS/MS analysis, to profile PYL5-interacting partners in plant cell lysates. With this strategy, three new PYL5-interacting partners, ubiquitin receptor RAD23C, COP9 signalosome complex subunit 1 (CSN1), and cyclase-associated protein 1 (CAP1), along with their key binding sites with PYL5 were identified. Among these proteins, CAP1 was verified to interact with PYL5 both in vitro and in vivo. The discovery of a new PYL5 binding partner showed the versatility of covalent chemical cross-linking and laid the foundation for future efforts to further elucidate the ABA signaling mechanism.

Foot-and-mouth disease virus (FMDV) exhibits broad antigenic diversity with poor intraserotype cross-neutralizing activity. Studies of the determinant involved in this diversity are essential for the development of broadly protective vaccines. In this work, we isolated a bovine antibody, designated R55, that displays cross-reaction with both FMDV A/AF/72 (hereafter named FMDV-AAF) and FMDV A/WH/09 (hereafter named FMDV-AWH) but only has a neutralizing effect on FMDV-AWH. Near-atomic resolution structures of FMDV-AAF-R55 and FMDV-AWH-R55 show that R55 engages the capsids of both FMDV-AAF and FMDV-AWH near the icosahedral 3-fold axis and binds to the βB and BC/HI-loops of VP2 and to the B-B knob of VP3. The common interaction residues are highly conserved, which is the major determinant for cross-reaction with both FMDV-AAF and FMDV-AWH. In addition, the cryo-EM structure of the FMDV-AWH-R55 complex also shows that R55 binds to (VP3)E70 located at the VP3 BC-loop in an adjacent pentamer, which enhances the acid and thermal stabilities of the viral capsid. This may prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection. In contrast, R55 binds only to the FMDV-AAF capsid within one pentamer due to the (VP3)E70G variation, which neither enhances capsid stability nor neutralizes FMDV-AAF infection. The (VP3)E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The crucial amino acid (VP3)E70 is a key component of the neutralizing epitopes, which may aid in the development of broadly protective vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISA) and virus neutralization tests (VNT) showed that R55 displays cross-reactions with both FMDV-AWH and FMDV-AAF but only has a neutralizing effect on FMDV-AWH. Cryo-EM structures, fluorescence-based thermal stability assays and acid stability assays showed that R55 engages the capsid of FMDV-AWH near the icosahedral 3-fold axis and informs an interpentamer epitope, which overstabilizes virions to hinder capsid dissociation to release the genome, eventually leading to neutralization of viral infection. The crucial amino acid (VP3)E70 forms a key component of neutralizing epitopes, and the determination of the (VP3)E70G mutation involved in the neutralizing differences between FMDV-AWH and FMDV-AAF could aid in the development of broadly protective vaccines.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that infects cloven-hoofed animals. Neutralizing antibodies play critical roles in antiviral infection. Although five known antigen sites that induce neutralizing antibodies have been defined, studies on cross-protective antigen sites are still scarce. We mapped two cross-protective antigen sites using 13 bovine-derived broadly neutralizing monoclonal antibodies (bnAbs) capable of neutralizing 4 lineages within 3 topotypes of FMDV serotype O. One antigen site was formed by a novel cluster of VP3-focused epitopes recognized by bnAb C4 and C4-like antibodies. The cryo-electron microscopy (cryo-EM) structure of the FMDV-OTi (O/Tibet/99)-C4 complex showed close contact with VP3 and a novel interprotomer antigen epitope around the icosahedral 3-fold axis of the FMDV particle, which is far beyond the known antigen site 4. The key determinants of the neutralizing function of C4 and C4-like antibodies on the capsid were βB (T65), the B-C loop (T68), the E-F loop (E131 and K134), and the H-I loop (G196), revealing a novel antigen site on VP3. The other antigen site comprised two group epitopes on VP2 recognized by 9 bnAbs (B57, B73, B77, B82, F28, F145, F150, E46, and E54), which belong to the known antigen site 2 of FMDV serotype O. Notably, bnAb C4 potently promoted FMDV RNA release in response to damage to viral particles, suggesting that the targeted epitope contains a trigger mechanism for particle disassembly. This study revealed two cross-protective antigen sites that can elicit cross-reactive neutralizing antibodies in cattle and provided new structural information for the design of a broad-spectrum molecular vaccine against FMDV serotype O. IMPORTANCE FMDV is the causative agent of foot-and-mouth disease (FMD), which is one of the most contagious and economically devastating diseases of domestic animals. The antigenic structure of FMDV serotype O is rather complicated, especially for those sites that can elicit a cross-protective neutralizing antibody response. Monoclonal neutralization antibodies provide both crucial defense components against FMDV infection and valuable tools for fine analysis of the antigenic structure. In this study, we found a cluster of novel VP3-focused epitopes using 13 bnAbs against FMDV serotype O from natural host cattle, which revealed two cross-protective antigen sites on VP2 and VP3. Antibody C4 targeting this novel epitope potently promoted viral particle disassembly and RNA release before infection, which may indicate a vulnerable region of FMDV. This study reveals new structural information about cross-protective antigen sites of FMDV serotype O, providing valuable and strong support for future research on broad-spectrum vaccines against FMD.

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV., Author summary FMDV is the causative agent of foot-and-mouth disease, one of the most contagious and economically devastating diseases of cloven-hoofed animals. The antigenic diversities of the currently known epitopes throughout FMDV serotypes and the lack of understanding of FMDV neutralization in natural hosts limit the development of a vaccine that is able to provide cross-serotype protection. In this work, we isolated FMDV serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) and a serotype O/A cross-reacting NAb (R50) from recovered natural bovine hosts and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. Structures of virus-NAb complex reveal F145 and B77 engage the capsid of FMDV-O near the icosahedral threefold axis. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes, revealing a previously unknown antigenic site. This is the first time to present structure details of FMDV neutralization by natural hosts. And this work also provides epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus (RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC-MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.

Adeno-associated virus (AAV) receptor (AAVR) is an essential receptor for the entry of multiple AAV serotypes with divergent rules; however, the mechanism remains unclear. Here, we determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, revealing the molecular details by which PKD1 recognizes AAV5 and PKD2 is solely engaged with AAV1. PKD2 lies on the plateau region of the AAV1 capsid. However, the AAV5-AAVR interface is strikingly different, in which PKD1 is bound at the opposite side of the spike of the AAV5 capsid than the PKD2-interacting region of AAV1. Residues in strands F/G and the CD loop of PKD1 interact directly with AAV5, whereas residues in strands B/C/E and the BC loop of PKD2 make contact with AAV1. These findings further the understanding of the distinct mechanisms by which AAVR recognizes various AAV serotypes and provide an example of a single receptor engaging multiple viral serotypes with divergent rules., Multiple adeno-associated viruses (AAV) use the same receptor (AAVR), but the binding mode is not clear. Here, the authors determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, identify residues necessary for virus entry and compare the receptor interfaces of different AAV capsids.

To date, an effective therapeutic treatment that confers strong attenuation toward coronaviruses (CoVs) remains elusive. Of all the potential drug targets, the helicase of CoVs is considered to be one of the most important. Here, we first present the structure of the full-length Nsp13 helicase of SARS-CoV (SARS-Nsp13) and investigate the structural coordination of its five domains and how these contribute to its translocation and unwinding activity. A translocation model is proposed for the Upf1-like helicase members according to three different structural conditions in solution characterized through H/D exchange assay, including substrate state (SARS-Nsp13-dsDNA bound with AMPPNP), transition state (bound with ADP-AlF4−) and product state (bound with ADP). We observed that the β19–β20 loop on the 1A domain is involved in unwinding process directly. Furthermore, we have shown that the RNA dependent RNA polymerase (RdRp), SARS-Nsp12, can enhance the helicase activity of SARS-Nsp13 through interacting with it directly. The interacting regions were identified and can be considered common across CoVs, which provides new insights into the Replication and Transcription Complex (RTC) of CoVs.

Viral infections are increasing and probably long-lasting global risks. In this study, a chemical library was exploited by phenotypic screening to discover new antiviral inhibitors. After optimizations from hit to lead, a novel potent small molecule (RYL-634) was identified, showing excellent broad-spectrum inhibition activity against various pathogenic viruses, including hepatitis C virus, dengue virus, Zika virus, chikungunya virus, enterovirus 71, human immunodeficiency virus, respiratory syncytial virus, and others. The mechanism of action and potential targets of RYL-634 were further explored by the combination of activity-based protein profiling and other techniques. Finally, human dihydroorotate dehydrogenase was validated as the major target of RYL-634. We did not observe any mutant resistance under our pressure selections with RYL-634, and it had a strong synergistic effect with some Food and Drug Administration-approved drugs. Hence, there is great potential for developing new broad-spectrum antivirals based on RYL-634.

Protein-protein interactions (PPIs) are indispensable in almost all cellular processes. Probing of complex PPIs provides new insights into the biological system of interest and paves the way for the development of therapeutics. Herein, we report a strategy for the capture of protein-protein interactions using photoaffinity palladium reagents. First, the palladium-mediated reagent site specifically transferred a photoaffinity modified aryl group to the designated cysteine residue. Next, the photoaffinity group was activated by UV radiation to trap the proximal protein residue for the formation of a crosslink. This strategy was used to capture the PYL-ABA-PP2C interaction, which is at the core of the abscisic acid (ABA) signalling pathway. Our results indicated that this palladium-mediated strategy can serve as an alternative for incorporating an increasing number of diverse substrates for protein crosslinking through cysteine modifications and can be explored for use in mapping protein-peptide or protein-protein interaction surfaces and in trapping potential interacting partners.

Summary Background The ongoing COVID-19 pandemic warrants accelerated efforts to test vaccine candidates. We aimed to assess the safety and immunogenicity of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate, BBIBP-CorV, in humans. Methods We did a randomised, double-blind, placebo-controlled, phase 1/2 trial at Shangqiu City Liangyuan District Center for Disease Control and Prevention in Henan Province, China. In phase 1, healthy people aged 18–80 years, who were negative for serum-specific IgM/IgG antibodies against SARS-CoV-2 at the time of screening, were separated into two age groups (18–59 years and ≥60 years) and randomly assigned to receive vaccine or placebo in a two-dose schedule of 2 μg, 4 μg, or 8 μg on days 0 and 28. In phase 2, healthy adults (aged 18–59 years) were randomly assigned (1:1:1:1) to receive vaccine or placebo on a single-dose schedule of 8 μg on day 0 or on a two-dose schedule of 4 μg on days 0 and 14, 0 and 21, or 0 and 28. Participants within each cohort were randomly assigned by stratified block randomisation (block size eight) and allocated (3:1) to receive vaccine or placebo. Group allocation was concealed from participants, investigators, and outcome assessors. The primary outcomes were safety and tolerability. The secondary outcome was immunogenicity, assessed as the neutralising antibody responses against infectious SARS-CoV-2. This study is registered with www.chictr.org.cn, ChiCTR2000032459. Findings In phase 1, 192 participants were enrolled (mean age 53·7 years [SD 15·6]) and were randomly assigned to receive vaccine (2 μg [n=24], 4 μg [n=24], or 8 μg [n=24] for both age groups [18–59 years and ≥60 years]) or placebo (n=24). At least one adverse reaction was reported within the first 7 days of inoculation in 42 (29%) of 144 vaccine recipients. The most common systematic adverse reaction was fever (18–59 years, one [4%] in the 2 μg group, one [4%] in the 4 μg group, and two [8%] in the 8 μg group; ≥60 years, one [4%] in the 8 μg group). All adverse reactions were mild or moderate in severity. No serious adverse event was reported within 28 days post vaccination. Neutralising antibody geometric mean titres were higher at day 42 in the group aged 18–59 years (87·7 [95% CI 64·9–118·6], 2 μg group; 211·2 [158·9–280·6], 4 μg group; and 228·7 [186·1–281·1], 8 μg group) and the group aged 60 years and older (80·7 [65·4–99·6], 2 μg group; 131·5 [108·2–159·7], 4 μg group; and 170·87 [133·0–219·5], 8 μg group) compared with the placebo group (2·0 [2·0–2·0]). In phase 2, 448 participants were enrolled (mean age 41·7 years [SD 9·9]) and were randomly assigned to receive the vaccine (8 μg on day 0 [n=84] or 4 μg on days 0 and 14 [n=84], days 0 and 21 [n=84], or days 0 and 28 [n=84]) or placebo on the same schedules (n=112). At least one adverse reaction within the first 7 days was reported in 76 (23%) of 336 vaccine recipients (33 [39%], 8 μg day 0; 18 [21%], 4 μg days 0 and 14; 15 [18%], 4 μg days 0 and 21; and ten [12%], 4 μg days 0 and 28). One placebo recipient in the 4 μg days 0 and 21 group reported grade 3 fever, but was self-limited and recovered. All other adverse reactions were mild or moderate in severity. The most common systematic adverse reaction was fever (one [1%], 8 μg day 0; one [1%], 4 μg days 0 and 14; three [4%], 4 μg days 0 and 21; two [2%], 4 μg days 0 and 28). The vaccine-elicited neutralising antibody titres on day 28 were significantly greater in the 4 μg days 0 and 14 (169·5, 95% CI 132·2–217·1), days 0 and 21 (282·7, 221·2–361·4), and days 0 and 28 (218·0, 181·8–261·3) schedules than the 8 μg day 0 schedule (14·7, 11·6–18·8; all p Interpretation The inactivated SARS-CoV-2 vaccine, BBIBP-CorV, is safe and well tolerated at all tested doses in two age groups. Humoral responses against SARS-CoV-2 were induced in all vaccine recipients on day 42. Two-dose immunisation with 4 μg vaccine on days 0 and 21 or days 0 and 28 achieved higher neutralising antibody titres than the single 8 μg dose or 4 μg dose on days 0 and 14. Funding National Program on Key Research Project of China, National Mega projects of China for Major Infectious Diseases, National Mega Projects of China for New Drug Creation, and Beijing Science and Technology Plan.

The Coronavirus Disease of 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health and economy. Therapeutic options such as monoclonal antibodies (mAbs) against SARS-CoV-2 are in urgent need. We have identified potent monoclonal antibodies binding to SARS-CoV-2 Spike protein from COVID-19 convalescent patients and one of these antibodies, P4A1, interacts directly and covers the majority of the Receptor Binding Motif (RBM) of Spike receptor-binding domain (RBD), shown by high-resolution complex structure analysis. We further demonstrated P4A1 binding and neutralizing activities against wild type and mutant spike proteins. P4A1 was subsequently engineered to reduce the potential risk for antibody-dependent enhancement (ADE) of infection and to extend its half-life. The engineered mAb exhibits optimized pharmacokinetic and safety profile, and results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection.

Summary Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre-/post- translocated polymerase complexes. The structures show notable structural rearrangements occurring to nsp12 and its cofactors nsp7/nsp8 to accommodate the nucleic acid compared to the apo complex, while there are highly conserved residues in nsp12 positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanisms of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription/replication machinery., Highlights • Structures of SARS-CoV-2 RNA polymerase in complexes with RNA revealed • Conformational changes in nsp8 and its interaction with the exiting RNA are observed • Incorporation and delayed-chain-termination mechanism of remdesivir is elucidated • Transition model from primase complex to polymerase complex is proposed, Cryo-EM structures of the SARS-CoV-2 RNA polymerase in complexes with RNA, before and after RNA translocation, reveals structural rearrangements that the RNA dependent RNA polymerase (RdRp) nsp12 and its cofactors (nsp7 and nsp8) undergo to accommodate nucleic acid binding. Further insights into how the complex is inhibited by remdesivir, and into the primase to polymerase transition, are also presented.

A novel coronavirus (2019-nCoV) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12), which catalyzes the synthesis of viral RNA, is a key component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. Here we report the cryo-EM structure of 2019-nCoV full-length nsp12 in complex with cofactors nsp7 and nsp8 at a resolution of 2.9-Å. Additional to the conserved architecture of the polymerase core of the viral polymerase family and a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain featured in coronaviral RdRp, nsp12 possesses a newly identified β-hairpin domain at its N-terminal. Key residues for viral replication and transcription are observed. A comparative analysis to show how remdesivir binds to this polymerase is also provided. This structure provides insight into the central component of coronaviral replication/transcription machinery and sheds light on the design of new antiviral therapeutics targeting viral RdRp.One Sentence SummaryStructure of 2019-nCov RNA polymerase.

Significance Mitochondria are dynamic double-membraned organelles, which undergo cycles of division and fusion mediated by dynamin-related GTPases. How dynamins mediate mitochondrial inner membrane fusion is not understood. In humans, defective inner membrane fusion causes a degenerative form of blindness termed dominant optic atrophy. Here, we solve the structure of a trimer assembly of an inner membrane fusion dynamin. Our observations underscore the structural plasticity of dynamins and suggest a previously unidentified mode of how they might mediate membrane fusion events., The fusion of inner mitochondrial membranes requires dynamin-like GTPases, Mgm1 in yeast and OPA1 in mammals, but how they mediate membrane fusion is poorly understood. Here, we determined the crystal structure of Saccharomyces cerevisiae short Mgm1 (s-Mgm1) in complex with GDP. It revealed an N-terminal GTPase (G) domain followed by two helix bundles (HB1 and HB2) and a unique C-terminal lipid-interacting stalk (LIS). Dimers can form through antiparallel HB interactions. Head-to-tail trimers are built by intermolecular interactions between the G domain and HB2-LIS. Biochemical and in vivo analyses support the idea that the assembly interfaces observed here are native and critical for Mgm1 function. We also found that s-Mgm1 interacts with negatively charged lipids via both the G domain and LIS. Based on these observations, we propose that membrane targeting via the G domain and LIS facilitates the in cis assembly of Mgm1, potentially generating a highly curved membrane tip to allow inner membrane fusion.

Abscisic acid (ABA) is a key phytohormone with diverse functions in plants, and its signal transduction is mainly mediated by ABA receptors termed PYR/PYL/RCARs (hereafter referred to as PYLs) through the PYLs-PP2Cs-SnRK2s regulatory systems. However, the model failed to account for the roles of some important known regulators of ABA physiology. Given the central role of PYLs in ABA signal transduction, we therefore speculated that ABA receptors PYLs might be involved in regulatory pathways other than PP2Cs. Thus, a comprehensive analysis of PYLs-interacting partners could greatly facilitate the identification of unknown regulatory pathways, advancing our knowledge of the ABA signaling mechanism. Herein, we present a strategy involving covalent chemical capture coupled with HPLC-MS/MS analysis, to profile PYL5-interacting partners in plant cell lysates. With this strategy, three new PYL5-interacting partners, ubiquitin receptor RAD23C, COP9 signalosome complex subunit 1 (CSN1), and cyclase-associated protein 1 (CAP1), along with their key binding sites with PYL5 were identified. Among these proteins, CAP1 was verified to interact with PYL5 both

Imaging live virus to monitor the viral entry process is essential to understand virus-host interactions during pathogen infection. However, methods for efficient labeling of live viruses, in particular labeling non-enveloped viruses and tracing virus entry processes, remain limited. Recently, labeling by using organometallic palladium reagents has provided a highly efficient and selective way to bioconjugate cysteines of virus proteins. Here, site-specific bioorthogonal labeling mediated by an organometallic palladium reagent on the surface of live enterovirus-71 (EV71) was used to visualize its entry into live cells. In contrast to currently used immunofluorescence and membrane-anchored dyes, this site-specific and quantitative labeling of live EV71 allows temporal imaging of its entry into host cell membranes on the timescale of seconds with little negative impact on its virulence. This method revealed details of EV71 virus entry and has broad applicability for monitoring virus entry that is difficult to assess by using conventional protein-labeling approaches.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Although several HCV protease/polymerase inhibitors were recently approved by U.S. FDA, the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistance. Viral entry is an essential target step for antiviral development, but FDA-approved HCV entry inhibitor remains exclusive. Here we identify serotonin 2A receptor (5-HT2AR) is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy. The silencing of 5-HT2AR and clinically available 5-HT2AR antagonist suppress cell culture-derived HCV (HCVcc) in different liver cells and primary human hepatocytes at late endocytosis process. The mechanism is related to regulate the correct plasma membrane localization of claudin 1 (CLDN1). Moreover, phenoxybenzamine (PBZ), an FDA-approved 5-HT2AR antagonist, inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs. The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice. Our results not only expand the understanding of HCV entry, but also present a promising target for the invention of HCV entry inhibitor. Electronic supplementary material The online version of this article (10.1007/s13238-018-0521-z) contains supplementary material, which is available to authorized users.