The potential for infection by coronaviruses (CoVs) has become a serious concern with the recent emergence of Middle East respiratory syndrome and severe acute respiratory syndrome (SARS) in the human population. CoVs encode two large polyproteins, which are then processed into 15–16 nonstructural proteins (nsps) that make significant contributions to viral replication and transcription by assembling the RNA replicase complex. Among them, nsp9 plays an essential role in viral replication by forming a homodimer that binds single‐stranded RNA. Thus, disrupting nsp9 dimerization is a potential anti‐CoV therapy. However, different nsp9 dimer forms have been reported for alpha‐ and beta‐CoVs, and no structural information is available for gamma‐CoVs. Here we determined the crystal structure of nsp9 from the avian infectious bronchitis virus (IBV), a representative gamma‐CoV that affects the economy of the poultry industry because it can infect domestic fowl. IBV nsp9 forms a homodimer via interactions across a hydrophobic interface, which consists of two parallel alpha helices near the carboxy terminus of the protein. The IBV nsp9 dimer resembles that of SARS‐CoV nsp9, indicating that this type of dimerization is conserved among all CoVs. This makes disruption of the dimeric interface an excellent strategy for developing anti‐CoV therapies. To facilitate this effort, we characterized the roles of six conserved residues on this interface using site‐directed mutagenesis and a multitude of biochemical and biophysical methods. We found that three residues are critical for nsp9 dimerization and its abitlity to bind RNA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of a potentially fatal disease named coronavirus disease 2019 (COVID-19), has raised significant public health concerns globally. To date, the COVID-19 pandemic has caused millions of people to be infected with SARS-CoV-2 worldwide. It has been known since the 2003 SARS epidemic that coronaviruses (CoVs) have large RNA genomes, the replication of which requires an RNA-dependent RNA replication/transcription complex. CoV nonstructural proteins (Nsps) play pivotal roles in the assembly of this complex and associated enzymatic functions in virus genomic replication. Several smaller nonenzymatic Nsps assist with RNA-dependent RNA polymerase function. In this study, we determined the structure of SARS-CoV-2 nonstructural protein 9 (nsp9), an RNA-binding protein that is essential for CoV replication. Its homotetrameric structure with two stable dimeric interfaces provids a structural basis for understanding the mechanisms of RNA-binding protein self-assembly, which may be essential for the regulation of viral RNA replication and transcription.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA could be detected in the blood of infected cases. From February 9, all blood establishments in Hubei province, China, implemented nucleic acid testing (NAT) for SARS‐CoV‐2 RNA among blood donors to ensure blood safety. Study Design and Methods Nucleic acid test screening individually (ID) or by minipool (MP) testing was performed according to the manufacturerʼs instructions. Inactivated culture supernatant of SARS‐CoV‐2–infected Vero cells was quantified by droplet digital polymerase chain reaction (ddPCR) and series diluted with negative plasma to evaluate the assayʼs performance. Results The limit of detection of the kit for MP testing was 62.94 and 33.14 copies/mL for N and ORF1ab region, respectively. ID testing could achieve 3.87 and 4.85 copies/mL for two regions using 1600 μL of plasma. Coefficients of variations of two different concentrations of reference samples were all less than 5% in MP testing. As of April 30, 2020, a total of 98,342 blood donations including 87,095 whole blood donations and 11,247 platelet donations were tested by ID or MP testing, and no RNAemia was found. In addition, Hubei province suffered precipitously decreased blood supply, especially in February: 86% reduction compared with the same period of 2019. Conclusion Nucleic acid test screening of SARS‐CoV‐2 on blood donations is suitable in blood establishments using the commercial real‐time PCR detection kit based on available instruments. The negative result indicated that SARS‐CoV‐2 appears to be no direct threat to blood safety but raises some serious issues for general blood supply.

Glycyrrhetinic acid (GA) is a natural product of licorice with mitochondria targeting properties and shows broad anticancer activities, but its targets and underlying mechanisms remain elusive. Here, we identified the mitochondrial enzyme serine hydroxymethyltransferase 2 (SHMT2) as a target of GA by using chemical proteomics. Binding to and inhibiting the activity of SHMT2 by GA were validated in vitro and in vivo. Knockout of SHMT2 or inhibiting SHMT2 with GA restricts mitochondrial energy supplies by downregulating mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid β-oxidation, and consequently suppresses cancer cell proliferation and tumor growth. Crystal structures of GA derivatives indicate that GA occupies SHMT2 folate-binding pocket and regulates SHMT2 activity. Modifications at GA carboxylic group with diamines significantly improved its anticancer potency, demonstrating GA as a decent structural template for SHMT2 inhibitor development.

Hepatitis A virus (HAV), an enigmatic and ancient pathogen, is a major causative agent of acute viral hepatitis worldwide. Although there are effective vaccines, antivirals against HAV infection are still required, especially during fulminant hepatitis outbreaks. A more in-depth understanding of the antigenic characteristics of HAV and the mechanisms of neutralization could aid in the development of rationally designed antiviral drugs targeting HAV. In this paper, 4 new antibodies—F4, F6, F7, and F9—are reported that potently neutralize HAV at 50% neutralizing concentration values (neut₅₀) ranging from 0.1 nM to 0.85 nM. High-resolution cryo-electron microscopy (cryo-EM) structures of HAV bound to F4, F6, F7, and F9, together with results of our previous studies on R10 fragment of antigen binding (Fab)-HAV complex, shed light on the locations and nature of the epitopes recognized by the 5 neutralizing monoclonal antibodies (NAbs). All the epitopes locate within the same patch and are highly conserved. The key structure-activity correlates based on the antigenic sites have been established. Based on the structural data of the single conserved antigenic site and key structure-activity correlates, one promising drug candidate named golvatinib was identified by in silico docking studies. Cell-based antiviral assays confirmed that golvatinib is capable of blocking HAV infection effectively with a 50% inhibitory concentration (IC₅₀) of approximately 1 μM. These results suggest that the single conserved antigenic site from complete HAV capsid is a good antiviral target and that golvatinib could function as a lead compound for anti-HAV drug development. Hepatitis A virus (HAV) is a unique, hepatotropic human picornavirus that infects approximately 1.5 million people annually and continues to cause mortality despite a successful vaccine. There are no licensed therapeutic drugs to date. Better knowledge of HAV antigenic features and neutralizing mechanisms will facilitate the development of HAV-targeting antiviral drugs. In this study, we report 4 potent HAV-specific neutralizing monoclonal antibodies (NAbs), together with our previous reported R10, that efficiently inhibit HAV infection by blocking attachment to the host cell. All 5 epitopes are located within the same patch and are highly conserved across 6 genotypes of human HAV, which suggests a single antigenic site for HAV, highlighting a prime target for structure-based drug design. Analysis of complexes with the 5 NAbs with varying neutralizing activities pinpointed key structure-activity correlates. By using a robust in silico docking method, one promising inhibitor named golvatinib was successfully identified from the DrugBank Database. In vitro assays confirmed its ability to block viral infection and revealed its neutralizing mechanism. Our approach could be useful in the design of effective drugs for picornavirus infections.