Efficacy and Safety of a Booster Vaccination with Two Inactivated SARS-CoV-2 Vaccines on Symptomatic COVID-19 Infection in Adults: Results of a Double-Blind, Randomized, Placebo-Controlled, Phase 3 Trial in Abu Dhabi.

IMPORTANCE: The protective efficacy of COVID-19 vaccinations has declined over time such that booster doses are required. OBJECTIVE: To evaluate the efficacy and adverse events of booster doses of two inactivated COVID-19 vaccines. DESIGN: This is a double-blind, randomized, placebo-controlled phase 3 trial aiming to evaluate the protective efficacy, safety, and immunogenicity of inactivated SARS-CoV-2 vaccine (Vero cells) after inoculation with booster doses of inactivated COVID-19 vaccine. SETTING: Healthy volunteers were recruited in an earlier phase 3 trial of two doses of inactivated vaccine. The participants in Abu Dhabi maintained the blind state of the trial and received a booster dose of vaccine or placebo at least six months after the primary immunization. PARTICIPANTS: Adults aged 18 and older with no history of SARS-CoV, SARS-CoV-2, or Middle East respiratory syndrome infection (via onsite inquiry) were screened for eligibility. INTERVENTIONS: A total of 9370 volunteers were screened and randomly allocated, of which 61 voluntarily withdrew from the screening stage without booster inoculation; 9309 received the booster vaccination, with 3083 in the WIV04 group, 3150 in the HB02 group, and 3076 in the alum-only group. Further, 5µg and 4µg of inactivated SARS-CoV-2 virion was adsorbed into aluminum hydroxide in a 0.5 mL aqueous suspension for WIV04 and HB02 vaccines. MAIN OUTCOMES AND MEASURES: The primary efficacy outcome was the prevention of PCR-confirmed symptomatic COVID-19 from 14 days after the booster vaccine in the per-protocol population. A safety analysis was performed in the intention-to-treat population. RESULTS: Symptomatic COVID-19 was identified in 36 participants in the WIV04 group (9.9 [95% CI, 7.2-13.8] per 1000 person-years), 28 in the HB02 group (7.6 [95% CI, 5.2-11.0] per 1000 person-years), and 193 in the alum-only group (55.2 [95% CI, 47.9-63.5] per 1000 person-years), resulting in a vaccine efficacy of 82.0% (95% CI, 74.2-87.8%) for WIV04 and 86.3% (95% CI, 79.6-91.1%) for HB02. One severe case of COVID-19 occurred in the alum-only group, and none occurred in the vaccine groups. Adverse reactions within seven days after vaccination occurred in 29.4% to 34.3% of participants in the three groups. Serious adverse events were rare and not related to vaccines (WIV04: 17 [0.5%]; HB02: 11 [0.4%]; alum only: 40 [1.3%]). CONCLUSIONS AND RELEVANCE: This study evaluated the safety of the booster dose, which was well tolerated by participants. Booster doses given over six months after the completion of primary immunization can help to provide more-effective protection against COVID-19 in healthy people 18 years of age or older. At the same time, the anti-SARS-CoV-2 antibodies produced by the two groups of experimental vaccines exhibited extensive cross-neutralization against representative SARS-CoV-2 variants. TRIAL REGISTRATION: This study is registered on ClinicalTrials.gov (NCT04510207).

A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors.

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.

A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analogue inhibitors

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 analogue 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. Graphical Structural analyses reveal how proteins from SARS-CoV-2 cooperate and use GTP to form the cap on viral mRNA, and how this process is interrupted by nucleotide analogues that serve as antiviral drugs.

Vaccination with Omicron Inactivated Vaccine in Pre-vaccinated Mice Protects against SARS-CoV-2 Prototype and Omicron Variants.

In response to the fast-waning immune response and the great threat of the Omicron variant of concern (VOC) to the public, we report the pilot-scale production of an inactivated Omicron vaccine candidate that induces high levels of neutralizing antibody titers to protect against the Omicron virus. Here, we demonstrate that the inactivated Omicron vaccine is safe and effective in recalling immune responses to the HB02, Omicron, and Delta viruses after one or two doses of BBIBP-CorV. In addition, the efficient productivity and good genetic stability of the manufactured inactivated vaccine is proved. These results support the further evaluation of the Omicron vaccine in a clinical trial.

Immunogenicity Evaluating of the Multivalent COVID-19 Inactivated Vaccine against the SARS-CoV-2 Variants.

It has been reported that the novel coronavirus (COVID-19) has caused more than 286 million cases and 5.4 million deaths to date. Several strategies have been implemented globally, such as social distancing and the development of the vaccines. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have appeared, such as Alpha, Beta, Gamma, Delta, and Omicron. With the rapid spread of the novel coronavirus and the rapidly changing mutants, the development of a broad-spectrum multivalent vaccine is considered to be the most effective way to defend against the constantly mutating virus. Here, we evaluated the immunogenicity of the multivalent COVID-19 inactivated vaccine. Mice were immunized by multivalent COVID-19 inactivated vaccine, and the neutralizing antibodies in serum were analyzed. The results show that HB02 + Delta + Omicron trivalent vaccine could provide broad spectrum protection against HB02, Beta, Delta, and Omicron virus. Additionally, the different multivalent COVID-19 inactivated vaccines could enhance cellular immunity. Together, our findings suggest that the multivalent COVID-19 inactivated vaccine can provide broad spectrum protection against HB02 and other virus variants in humoral and cellular immunity, providing new ideas for the development of a broad-spectrum COVID-19 vaccine.

The Life of SARS-CoV-2 Inside Cells: Replication-Transcription Complex Assembly and Function.

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.

Safety and immunogenicity of an inactivated COVID-19 vaccine, BBIBP-CorV, in people younger than 18 years: a randomised, double-blind, controlled, phase 1/2 trial.

BACKGROUND: Although SARS-CoV-2 infection often causes milder symptoms in children and adolescents, young people might still play a key part in SARS-CoV-2 transmission. An efficacious vaccine for children and adolescents could therefore assist pandemic control. For further evaluation of the inactivated COVID-19 vaccine candidate BBIBP-CorV, we assessed the safety and immunogenicity of BBIBP-CorV in participants aged 3-17 years. METHODS: A randomised, double-blind, controlled, phase 1/2 trial was done at Shangqiu City Liangyuan District Center for Disease Control and Prevention in Henan, China. In phases 1 and 2, healthy participants were stratified according to age (3-5 years, 6-12 years, or 13-17 years) and dose group. Individuals with a history of SARS-CoV-2 or SARS-CoV infection were excluded. All participants were randomly assigned, using stratified block randomisation (block size eight), to receive three doses of 2 µg, 4 µg, or 8 µg of vaccine or control (1:1:1:1) 28 days apart. The primary outcome, safety, was analysed in the safety set, which consisted of participants who had received at least one vaccination after being randomly assigned, and had any safety evaluation information. The secondary outcomes were geometric meant titre (GMT) of the neutralising antibody against infectious SARS-CoV-2 and were analysed based on the full analysis set. This study is registered with www.chictr.org.cn, ChiCTR2000032459, and is ongoing. FINDINGS: Between Aug 14, 2020, and Sept 24, 2020, 445 participants were screened, and 288 eligible participants were randomly assigned to vaccine (n=216, 24 for each dose level [2/4/8 µg] in each of three age cohorts [3-5, 6-12, and 13-17 years]) or control (n=72, 24 for each age cohort [3-5, 6-12, and 13-17 years]) in phase 1. In phase 2, 810 participants were screened and 720 eligible participants were randomly assigned and allocated to vaccine (n=540, 60 for each dose level [2/4/8 µg] in each of three age cohorts [3-5, 6-12, and 13-17 years]) or control (n=180, 60 for each age cohort [3-5, 6-12, and 13-17 years]). The most common injection site adverse reaction was pain (ten [4%] 251 participants in all vaccination groups of the 3-5 years cohort; 23 [9·1%] of 252 participants in all vaccination groups and one [1·2%] of 84 in the control group of the 6-12 years cohort; 20 [7·9%] of 252 participants in all vaccination groups of the 13-17 years cohort). The most common systematic adverse reaction was fever (32 [12·7%] of 251 participants in all vaccination groups and six [7·1%] of 84 participants in the control group of the 3-5 years cohort; 13 [5·2%] of 252 participants in the vaccination groups and one [1·2%] of 84 in the control group of the 6-12 years cohort; 26 [10·3%] of 252 participants in all vaccination groups and eight [9·5%] of 84 in the control group of the 13-17 years cohort). Adverse reactions were mostly mild to moderate in severity. The neutralising antibody GMT against the SARS-CoV-2 virus ranged from 105·3 to 180·2 in the 3-5 years cohort, 84·1 to 168·6 in the 6-12 years cohort, and 88·0 to 155·7 in the 13-17 years cohort on day 28 after the second vaccination; and ranged from 143·5 to 224·4 in the 3-5 years cohort, 127 to 184·8 in the 6-12 years cohort, and 150·7 to 199 in the 13-17 years cohort on day 28 after the third vaccination. INTERPRETATION: The inactivated COVID-19 vaccine BBIBP-CorV is safe and well tolerated at all tested dose levels in participants aged 3-17 years. BBIBP-CorV also elicited robust humoral responses against SARS-CoV-2 infection after two doses. Our findings support the use of a 4 µg dose and two-shot regimen BBIBP-CorV in phase 3 trials in the population younger than 18 years to further ascertain its safety and protection efficacy against COVID-19. 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. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

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

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-nsp82-nsp12-nsp132-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.

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

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.

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

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.

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

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) (7MeGpppA) 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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 ß-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.