Publications by authors named "Zhiyong Lou"

145 Publications

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

Cell 2021 06 24;184(13):3474-3485.e11. Epub 2021 May 24.

MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China; Guangzhou Laboratory, Guangzhou, China. Electronic address:

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (GpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.
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http://dx.doi.org/10.1016/j.cell.2021.05.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142856PMC
June 2021

Rising to the challenge of COVID-19: Working on SARS-CoV-2 during the pandemic.

Mol Cell 2021 06;81(11):2261-2265

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.
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http://dx.doi.org/10.1016/j.molcel.2021.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173667PMC
June 2021

A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes.

Nat Commun 2021 05 11;12(1):2623. Epub 2021 May 11.

Shanghai Synchrotron Radiation Facility, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, People's Republic of China.

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.
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http://dx.doi.org/10.1038/s41467-021-22926-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113581PMC
May 2021

Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes.

PLoS Biol 2021 05 7;19(5):e3001209. Epub 2021 May 7.

MOE Key Laboratory of Protein Science & Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, China.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.
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http://dx.doi.org/10.1371/journal.pbio.3001209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133496PMC
May 2021

Reply to: Errors in the deposited SFTSV L protein structure.

Nat Microbiol 2021 May 29;6(5):551-552. Epub 2021 Apr 29.

MOE Key Laboratory of Protein Sciences, School of Medicine, Tsinghua University, Beijing, China.

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http://dx.doi.org/10.1038/s41564-021-00902-2DOI Listing
May 2021

Structures of Foot-and-mouth Disease Virus with neutralizing antibodies derived from recovered natural host reveal a mechanism for cross-serotype neutralization.

PLoS Pathog 2021 04 28;17(4):e1009507. Epub 2021 Apr 28.

MOE Key Laboratory of Protein Science & Collaborative Innovation Center of Biotherapy, School of Medicine and School of Life Sciences, Tsinghua University, Beijing, China.

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.
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http://dx.doi.org/10.1371/journal.ppat.1009507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081260PMC
April 2021

Author Correction: Structure of severe fever with thrombocytopenia syndrome virus L protein elucidates the mechanisms of viral transcription initiation.

Nat Microbiol 2021 May;6(5):697-698

MOE Key Laboratory of Protein Science and Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, China.

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http://dx.doi.org/10.1038/s41564-021-00906-yDOI Listing
May 2021

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Cell 2021 01 14;184(1):184-193.e10. Epub 2020 Nov 14.

MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China. Electronic address:

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp8-nsp12-nsp13-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.
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http://dx.doi.org/10.1016/j.cell.2020.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666536PMC
January 2021

Architecture of a SARS-CoV-2 mini replication and transcription complex.

Nat Commun 2020 11 18;11(1):5874. Epub 2020 Nov 18.

MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China.

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.
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http://dx.doi.org/10.1038/s41467-020-19770-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675986PMC
November 2020

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBIBP-CorV: a randomised, double-blind, placebo-controlled, phase 1/2 trial.

Lancet Infect Dis 2021 01 15;21(1):39-51. Epub 2020 Oct 15.

Chinese Center for Disease Control and Prevention, Beijing, China.

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<0·001).

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.
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http://dx.doi.org/10.1016/S1473-3099(20)30831-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561304PMC
January 2021

Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2.

Cell 2020 08 6;182(3):713-721.e9. Epub 2020 Jun 6.

National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China. Electronic address:

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 μg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.
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http://dx.doi.org/10.1016/j.cell.2020.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275151PMC
August 2020

Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Cell 2020 07 22;182(2):417-428.e13. Epub 2020 May 22.

Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China; Laboratory of Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China; National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, CAS, Beijing, China. Electronic address:

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- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas 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 mechanism 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 and replication machinery.
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http://dx.doi.org/10.1016/j.cell.2020.05.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242921PMC
July 2020

Structural insights into G domain dimerization and pathogenic mutation of OPA1.

J Cell Biol 2020 07;219(7)

College of Life Sciences and State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China.

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF3-, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.
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http://dx.doi.org/10.1083/jcb.201907098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337494PMC
July 2020

Structure of severe fever with thrombocytopenia syndrome virus L protein elucidates the mechanisms of viral transcription initiation.

Nat Microbiol 2020 06 27;5(6):864-871. Epub 2020 Apr 27.

MOE Key Laboratory of Protein Science and Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, China.

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 Å, 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.
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http://dx.doi.org/10.1038/s41564-020-0712-2DOI Listing
June 2020

Infectious Disease Research in China.

Authors:
Zhiyong Lou

ACS Infect Dis 2020 05 24;6(5):760. Epub 2020 Apr 24.

Tsinghua University School of Medicine, Beijing 100084, China.

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http://dx.doi.org/10.1021/acsinfecdis.0c00220DOI Listing
May 2020

Structure of the RNA-dependent RNA polymerase from COVID-19 virus.

Science 2020 05 10;368(6492):779-782. Epub 2020 Apr 10.

Laboratory of Structural Biology, School of Life Sciences, and School of Medicine, Tsinghua University, Beijing, China.

A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified β-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.
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http://dx.doi.org/10.1126/science.abb7498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164392PMC
May 2020

Structural analysis of a trimeric assembly of the mitochondrial dynamin-like GTPase Mgm1.

Proc Natl Acad Sci U S A 2020 02 10;117(8):4061-4070. Epub 2020 Feb 10.

National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China;

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 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.
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http://dx.doi.org/10.1073/pnas.1919116117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049166PMC
February 2020

Identification of Interferon Receptor IFNAR2 As a Novel HCV Entry Factor by Using Chemical Probes.

ACS Chem Biol 2020 05 3;15(5):1232-1241. Epub 2020 Feb 3.

MOE Key Laboratory of Protein Science and Collaborative Innovation Center for Biotherapy, School of Medicine, Tsinghua University, Beijing, 100084, China.

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 . 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.
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http://dx.doi.org/10.1021/acschembio.9b00912DOI Listing
May 2020

Novel strategy for expression and characterization of rabies virus glycoprotein.

Protein Expr Purif 2020 04 3;168:105567. Epub 2020 Jan 3.

AnyGo Technology, Beijing, PR China; AbMax Biotechnology, Beijing, PR China. Electronic address:

Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few μg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection.
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http://dx.doi.org/10.1016/j.pep.2019.105567DOI Listing
April 2020

Discovery of Interacting Proteins of ABA Receptor PYL5 Covalent Chemical Capture.

ACS Chem Biol 2019 12 22;14(12):2557-2563. Epub 2019 Oct 22.

School of Medicine , Tsinghua University , Beijing 100084 , China.

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 and . 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.
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http://dx.doi.org/10.1021/acschembio.9b00806DOI Listing
December 2019

Activity-Based Protein Profiling Identifies ATG4B as a Key Host Factor for Enterovirus 71 Proliferation.

J Virol 2019 12 26;93(24). Epub 2019 Nov 26.

Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, China

Virus-encoded proteases play diverse roles in the efficient replication of enterovirus 71 (EV71), which is the causative agent of human hand, foot, and mouth disease (HFMD). However, it is unclear how host proteases affect viral proliferation. Here, we designed activity-based probes (ABPs) based on an inhibitor of the main EV71 protease (3C), which is responsible for the hydrolysis of the EV71 polyprotein, and successfully identified host candidates that bind to the ABPs. Among the candidates, the host cysteine protease autophagy-related protein 4 homolog B (ATG4B), a key component of the autophagy machinery, was demonstrated to hydrolytically process the substrate of EV71 3C and had activity comparable to that of the viral protease. Genetic disruption of ATG4B confirmed that the enzyme is indispensable for viral proliferation Our results not only further the understanding of host-virus interactions in EV71 biology but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions. Enterovirus 71 (EV71), one of the major pathogens of human HFMD, has caused outbreaks worldwide. How EV71 efficiently assesses its life cycle with elaborate interactions with multiple host factors remains to be elucidated. In this work, we deconvoluted that the host ATG4B protein processes the viral polyprotein with its cysteine protease activity and helps EV71 replicate through a chemical biology strategy. Our results not only further the understanding of the EV71 life cycle but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.
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http://dx.doi.org/10.1128/JVI.01092-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880168PMC
December 2019

Divergent engagements between adeno-associated viruses with their cellular receptor AAVR.

Nat Commun 2019 08 21;10(1):3760. Epub 2019 Aug 21.

MOE Key Laboratory of Protein Science & Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, 100084, China.

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.
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http://dx.doi.org/10.1038/s41467-019-11668-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704107PMC
August 2019

Proteomic Profiling of Purified Rabies Virus Particles.

Virol Sin 2020 Apr 19;35(2):143-155. Epub 2019 Aug 19.

Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Changchun, 130122, China.

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.
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http://dx.doi.org/10.1007/s12250-019-00157-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090697PMC
April 2020

Photoaffinity palladium reagents for capture of protein-protein interactions.

Org Biomol Chem 2019 07 19;17(26):6369-6373. Epub 2019 Jun 19.

Collaborative Innovation Center of Biotherapy and MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing 100084, China.

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.
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http://dx.doi.org/10.1039/c9ob01048cDOI Listing
July 2019

Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis.

Nucleic Acids Res 2019 07;47(12):6538-6550

Laboratory of Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China.

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.
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http://dx.doi.org/10.1093/nar/gkz409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614802PMC
July 2019

Discovery, Optimization, and Target Identification of Novel Potent Broad-Spectrum Antiviral Inhibitors.

J Med Chem 2019 04 12;62(8):4056-4073. Epub 2019 Apr 12.

College of Life Sciences , Hebei Normal University , Shijiazhuang , Hebei 050024 , P. R. China.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00091DOI Listing
April 2019

Adeno-associated virus 2 bound to its cellular receptor AAVR.

Nat Microbiol 2019 04 11;4(4):675-682. Epub 2019 Feb 11.

MOE Laboratory of Protein Science and Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, China.

Adeno-associated virus (AAV) is a leading vector for virus-based gene therapy. The receptor for AAV (AAVR; also named KIAA0319L) was recently identified, and the precise characterization of AAV-AAVR recognition is in immediate demand. Taking advantage of a particle-filtering algorithm, we report here the cryo-electron microscopy structure of the AAV2-AAVR complex at 2.8 Å resolution. This structure reveals that of the five Ig-like polycystic kidney disease (PKD) domains in AAVR, PKD2 binds directly to the spike region of the AAV2 capsid adjacent to the icosahedral three-fold axis. Residues in strands B and E, and the BC loop of AAVR PKD2 interact directly with the AAV2 capsid. The interacting residues in the AAV2 capsid are mainly in AAV-featured variable regions. Mutagenesis of the amino acids at the AAV2-AAVR interface reduces binding activity and viral infectivity. Our findings provide insights into the biology of AAV entry with high-resolution details, providing opportunities for the development of new AAV vectors for gene therapy.
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http://dx.doi.org/10.1038/s41564-018-0356-7DOI Listing
April 2019

Seneca Valley virus attachment and uncoating mediated by its receptor anthrax toxin receptor 1.

Proc Natl Acad Sci U S A 2018 12 4;115(51):13087-13092. Epub 2018 Dec 4.

Drug Discovery Center for Infectious Disease, College of Pharmacy, Nankai University, 300071 Tianjin, China.

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. SVV mediates cell entry by attachment to the receptor anthrax toxin receptor 1 (ANTXR1). Here we determine atomic structures of mature SVV particles alone and in complex with ANTXR1 in both neutral and acidic conditions, as well as empty "spent" particles in complex with ANTXR1 in acidic conditions by cryoelectron microscopy. SVV engages ANTXR1 mainly by the VP2 DF and VP1 CD loops, leading to structural changes in the VP1 GH loop and VP3 GH loop, which attenuate interprotomer interactions and destabilize the capsid assembly. Despite lying on the edge of the attachment site, VP2 D146 interacts with the metal ion in ANTXR1 and is required for cell entry. Though the individual substitution of most interacting residues abolishes receptor binding and virus propagation, a serine-to-alanine mutation at VP2 S177 significantly increases SVV proliferation. Acidification of the SVV-ANTXR1 complex results in a major reconfiguration of the pentameric capsid assemblies, which rotate ∼20° around the icosahedral fivefold axes to form a previously uncharacterized spent particle resembling a potential uncoating intermediate with remarkable perforations at both two- and threefold axes. These structures provide high-resolution snapshots of SVV entry, highlighting opportunities for anticancer therapeutic optimization.
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http://dx.doi.org/10.1073/pnas.1814309115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304951PMC
December 2018

An electron transfer path connects subunits of a mycobacterial respiratory supercomplex.

Science 2018 11 25;362(6418). Epub 2018 Oct 25.

State Key Laboratory of Medicinal Chemical Biology and College of Life Science, Nankai University, Tianjin 300353, China.

We report a 3.5-angstrom-resolution cryo-electron microscopy structure of a respiratory supercomplex isolated from It comprises a complex III dimer flanked on either side by individual complex IV subunits. Complex III and IV associate so that electrons can be transferred from quinol in complex III to the oxygen reduction center in complex IV by way of a bridging cytochrome subunit. We observed a superoxide dismutase-like subunit at the periplasmic face, which may be responsible for detoxification of superoxide formed by complex III. The structure reveals features of an established drug target and provides a foundation for the development of treatments for human tuberculosis.
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http://dx.doi.org/10.1126/science.aat8923DOI Listing
November 2018
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