Yang Han

Bio: Yang Han is an academic researcher from Chinese Academy of Sciences. The author has contributed to research in topics: Medicine & RNA. The author has an hindex of 5, co-authored 11 publications receiving 12716 citations. Previous affiliations of Yang Han include Wuhan Jinyintan Hospital.

The ORF3a protein of SARS-CoV-2 induces apoptosis in cells.

Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the ongoing pandemic of Coronavirus Disease 2019. SARS-CoV-2 belongs to the genus Betacoronavirus of the Coronaviridae family, which includes SARS-CoV and Middle East respiratory syndrome coronavirus. Coronavirus-encoded accessory proteins play critical roles in virus–host interactions and the modulation of host immune responses, thereby contributing to coronaviral pathogenicity via different strategies. However, the functions of SARS-CoV-2-encoded accessory proteins are not well understood. Apoptosis is a predominant type of programmed cell death, and has been recognized as an important host antiviral defense mechanism that controls viral infection and regulates the inflammatory response. Previous studies have reported that the SARS-CoV-encoded accessory protein ORF3a can induce apoptosis in cells, leading to the question of whether SARS-CoV-2 ORF3a also has pro-apoptotic activity. Here, we investigated the potential apoptosis-inducing activity of SARS-CoV-2 ORF3a in different cell lines and compared the pro-apoptotic activities of SARS-CoV-2 ORF3a with those of SARS-CoV ORF3a using the same system. We sought to determine whether SARS-CoV-2 ORF3a can induce apoptosis using annexin V-fluorescein 5-isothiocyanate(FITC)/propidium iodide (PI) double staining in cultured HEK293T, HepG2, and Vero E6 cells. We found that annexin V and PI staining was significantly increased in cells expressing SARS-CoV-2 ORF3a compared with that in control cells (Fig. 1a). Moreover, the quantified data based on measuring the apoptosis rate also confirmed the pro-apoptotic activity of ORF3a in different cell lines (Fig. 1b). Furthermore, we examined activated caspase-3, a marker of caspase-dependent apoptosis, by flow cytometry and found that the percentage of cells with activated caspase-3 was significantly elevated in the presence of ORF3a (Fig. 1c). These results show that SARS-CoV-2 ORF3a can efficiently induce apoptosis in cells. To determine the mechanism through which SARS-CoV-2 ORF3a induces apoptosis, activation of the apoptosis cascade in HEK293T cells expressing ORF3a was examined by western blotting, probing for some apoptosis pathway components at 24 and 48 h post transfection. Cells treated with staurosporine, an apoptosis inducer, were used as a positive control. SARS-CoV-2 ORF3a induced the cleavage/activation of caspase-8, whereas Bcl-2 expression levels were not affected (Fig. 1d). The cleavage/activation of caspase-8 is recognized as a hallmark of the extrinsic apoptotic pathway, whereas Bcl-2 plays an important role in initiation of the intrinsic pathway. Moreover, we found that the levels of truncated Bid (tBid), cleaved caspase-9, and cytochrome c were elevated in the presence of SARS-CoV-2 ORF3a (Fig. 1e), and either a caspase-8 or caspase-9 inhibitor significantly suppressed SARS-CoV-2 ORF3a-induced apoptosis (Fig. 1f, g). Thus, our results imply that SARS-CoV-2 ORF3a can induce apoptosis via the extrinsic pathway, in which activated caspase-8 cleaves Bid to tBid and in turn induces the release of mitochondrial cytochrome c, resulting in apoptosome formation and caspase-9 cleavage/activation. We next sought to examine the relationship between the membrane association and pro-apoptotic activity of SARS-CoV-2 ORF3a. As previously reported, SARS-CoV ORF3a is a transmembrane protein that contains several conserved motifs including a cysteine-rich motif (a.a.127–133), tyrosine-based sorting motif (YXXΦ; a.a.160–163), and diacidic EXD motif (a.a. 171–173), and these domains regulate the subcellular location of SARS-CoV ORF3a and play important roles in SARS-CoV ORF3a infection, inducing apoptosis. SARS-CoV-2 ORF3a shares 73% amino acid homology with its counterpart in SARS-CoV, and the cysteine-rich and YXXΦ motifs are conserved but the EXD motif was found to be changed to SGD in SARS-CoV-2 ORF3a (Fig. S1a). Thus, we constructed two mutant ORF3a proteins by mutating C130/133 of the cysteine-rich motif to S (SARS-CoV-2 ORF3a-CS) or Y160 of the YXXΦ motif to A (SARS-CoV-2 ORF3a-YA). The immunofluorescence assays showed that wild-type ORF3a of SARS-CoV-2 (ORF3a-WT) localized to the plasma membrane with punctate cytoplasmic staining, whereas ORF3a-CS and ORF3a-YA exhibited more cytoplasmic localization (Figs. 1h and S1b). The results of cytosol-membrane fractionation assays showed that whereas ORF3a-WT was present in both cytosol and membrane fractions, either ORF3a-CS or ORF3a-YA was absent in the membrane fraction (Figs. 1i and S1c). Moreover, we found that ORF3a-CS or ORF3a-YA showed minimal apoptosis-inducing and caspase-3activiting activity in cells in the presence or absence of z-VAD-fmk, a general caspase inhibitor (Figs. 1j and S1d). In addition, ORF3aCS or ORF3a-YA failed to induce the cleavage of Bid, caspase-8, and caspase-9 or the release of cytochrome c (Fig.1k, l). These results indicate that membrane association is required for the proapoptotic activity of SARS-CoV-2 ORF3a. To investigate if there is any difference between the proapoptotic activities of ORF3a proteins of SARS-CoV-2 and SARS-CoV, we examined the membrane association and apoptosis-induction ability of SARS-CoV ORF3a. SARS-CoV ORF3a variants were generated by mutating C127/130/133 to S (SARS-CoV ORF3a-CS)

Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a

Abstract: CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.

Clinical Characteristics of Coronavirus Disease 2019 in China.

Abstract: Background Since December 2019, when coronavirus disease 2019 (Covid-19) emerged in Wuhan city and rapidly spread throughout China, data have been needed on the clinical characteristics of...

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China.

Abstract: Importance In December 2019, novel coronavirus (2019-nCoV)–infected pneumonia (NCIP) occurred in Wuhan, China. The number of cases has increased rapidly but information on the clinical characteristics of affected patients is limited. Objective To describe the epidemiological and clinical characteristics of NCIP. Design, Setting, and Participants Retrospective, single-center case series of the 138 consecutive hospitalized patients with confirmed NCIP at Zhongnan Hospital of Wuhan University in Wuhan, China, from January 1 to January 28, 2020; final date of follow-up was February 3, 2020. Exposures Documented NCIP. Main Outcomes and Measures Epidemiological, demographic, clinical, laboratory, radiological, and treatment data were collected and analyzed. Outcomes of critically ill patients and noncritically ill patients were compared. Presumed hospital-related transmission was suspected if a cluster of health professionals or hospitalized patients in the same wards became infected and a possible source of infection could be tracked. Results Of 138 hospitalized patients with NCIP, the median age was 56 years (interquartile range, 42-68; range, 22-92 years) and 75 (54.3%) were men. Hospital-associated transmission was suspected as the presumed mechanism of infection for affected health professionals (40 [29%]) and hospitalized patients (17 [12.3%]). Common symptoms included fever (136 [98.6%]), fatigue (96 [69.6%]), and dry cough (82 [59.4%]). Lymphopenia (lymphocyte count, 0.8 × 109/L [interquartile range {IQR}, 0.6-1.1]) occurred in 97 patients (70.3%), prolonged prothrombin time (13.0 seconds [IQR, 12.3-13.7]) in 80 patients (58%), and elevated lactate dehydrogenase (261 U/L [IQR, 182-403]) in 55 patients (39.9%). Chest computed tomographic scans showed bilateral patchy shadows or ground glass opacity in the lungs of all patients. Most patients received antiviral therapy (oseltamivir, 124 [89.9%]), and many received antibacterial therapy (moxifloxacin, 89 [64.4%]; ceftriaxone, 34 [24.6%]; azithromycin, 25 [18.1%]) and glucocorticoid therapy (62 [44.9%]). Thirty-six patients (26.1%) were transferred to the intensive care unit (ICU) because of complications, including acute respiratory distress syndrome (22 [61.1%]), arrhythmia (16 [44.4%]), and shock (11 [30.6%]). The median time from first symptom to dyspnea was 5.0 days, to hospital admission was 7.0 days, and to ARDS was 8.0 days. Patients treated in the ICU (n = 36), compared with patients not treated in the ICU (n = 102), were older (median age, 66 years vs 51 years), were more likely to have underlying comorbidities (26 [72.2%] vs 38 [37.3%]), and were more likely to have dyspnea (23 [63.9%] vs 20 [19.6%]), and anorexia (24 [66.7%] vs 31 [30.4%]). Of the 36 cases in the ICU, 4 (11.1%) received high-flow oxygen therapy, 15 (41.7%) received noninvasive ventilation, and 17 (47.2%) received invasive ventilation (4 were switched to extracorporeal membrane oxygenation). As of February 3, 47 patients (34.1%) were discharged and 6 died (overall mortality, 4.3%), but the remaining patients are still hospitalized. Among those discharged alive (n = 47), the median hospital stay was 10 days (IQR, 7.0-14.0). Conclusions and Relevance In this single-center case series of 138 hospitalized patients with confirmed NCIP in Wuhan, China, presumed hospital-related transmission of 2019-nCoV was suspected in 41% of patients, 26% of patients received ICU care, and mortality was 4.3%.

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.

Abstract: The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.