Engineering customized viral receptors for various coronaviruses (preprint)

Coronaviruses display versatile receptor usage, yet in-depth characterization of coronaviruses lacking known receptor identities has been impeded by the absence of feasible infection models. Here, we developed an innovative strategy to engineer functional customized viral receptors (CVRs). The modular design relies on building receptor frameworks comprising various function modules and generating specific epitope-targeting viral binding domains. We showed the key factors for CVRs to efficiently facilitate spike cleavage, membrane fusion, pseudovirus entry, and authentic virus amplification for various coronaviruses, resembling their native receptors. Applying this strategy, we delineated the accessible receptor binding epitopes for functional SARS-CoV-2 CVR design and elucidated the mechanism of entry supported by an amino-terminus domain (NTD) targeting S2L20-CVR. Furthermore, we created CVR-expressing cells for assessing antibodies and inhibitors against 12 representative coronaviruses from six subgenera, most of which lacking known receptors. Notably, a pan-sarbecovirus CVR supported entry of various sarbecoviruses, as well as amplification of a replicable HKU3 pseudovirus and the authentic strain RsHuB2019A. Through combining an HKU5-specific CVR with reverse genetics, we successfully rescued and cultured wild-type and fluorescence protein-incorporated HKU5, a receptor-unidentified merbecovirus. Our study demonstrated the great potential of CVR strategy in establishing native receptor-independent infection models, paving the way for studying various viruses that are challenging to culture due to the lack of susceptible cells.

Engineering customized viral receptors for various coronaviruses (preprint)

Coronaviruses display versatile receptor usage, yet in-depth characterization of coronaviruses lacking known receptor identities has been impeded by the absence of feasible infection models1,2. Here, we developed an innovative strategy to engineer functional customized viral receptors (CVRs). The modular design relies on building receptor frameworks comprising various function modules and generating specific epitope-targeting viral binding domains. We showed the key factors for CVRs to efficiently facilitate spike cleavage, membrane fusion, pseudovirus entry, and authentic virus propagation for various coronaviruses, resembling their native receptors. Applying this strategy, we delineated the accessible receptor binding epitopes for functional SARS-CoV-2 CVR design and elucidated the mechanism of entry supported by an amino-terminus domain (NTD) targeting S2L20-CVR. Furthermore, we created CVR-expressing cells for assessing antibodies and inhibitors against 12 representative coronaviruses from six subgenera, most of which lacking known receptors. Notably, a pan-sarbecovirus CVR supported entry of various sarbecoviruses, as well as propagation of a replicable HKU3 pseudovirus and the authentic strain RsHuB2019A3. Through combining an HKU5-specific CVR with reverse genetics, we successfully rescued and cultured wild-type and fluorescence protein-incorporated HKU5, a receptor-unidentified merbecovirus. Our study demonstrated the great potential of CVR strategy in establishing native receptor-independent infection models, paving the way for studying various viruses that are challenging to culture due to the lack of susceptible cells.

Doctors' and Patients' Perceptions of Impacts of Doctors' Communication and Empathy Skills on Doctor-Patient Relationships During COVID-19.

BACKGROUND: During the COVID-19 pandemic, the performance of Chinese doctors may have led to improved doctor-patient relationships (DPRs). However, it is unclear how doctors and patients perceived the impact of doctors' communication and empathy skills on DPRs during the COVID-19 pandemic. OBJECTIVE: To examine the perceptions of doctors and patients on how doctors' communication skills and empathy skills influence DPRs during COVID-19. MAIN MEASURES: Doctors' and patients' perceptions of doctors' communication skills were measured using the Chinese version of the SEGUE Framework. To measure empathy skills and DPRs, the Jefferson Scale of Empathy and Difficult Doctor-Patient Relationship Questionnaire were administered to doctors, and the Consultation and Relational Empathy Measure and Patient-Doctor Relationship Questionnaire were administered to patients. RESULTS: A total of 902 doctors and 1432 patients in China were recruited during the pandemic via online or offline surveys (overall response rate of 69.8%). Both doctors and patients rated doctors' empathy skills as more impactful on DPRs than communication skills. Doctors believed that only their empathy skills influenced DPRs. But patients believed that there was a significant bi-directional relationship between doctors' communication and empathy skills and these two skills interacted to directly and indirectly influence DPRs, and doctors' empathy had a greater mediating effect than their communication. CONCLUSIONS: During COVID-19, there were both similarities and differences between Chinese doctors' and patients' views on how doctors' communication and empathy skills influenced DPRs. The greater effect of doctors' empathy skills suggests that both doctors and patients attach more importance to doctors' empathy in doctor-patient interactions. The bi-directional effect on patient outcomes suggests that both doctors' communication and empathy skills are important to patients' perceptions of DPRs.

Doctors’ and Patients’ Perceptions of Impacts of Doctors’ Communication and Empathy Skills on Doctor–Patient Relationships During COVID-19

Background During the COVID-19 pandemic, the performance of Chinese doctors may have led to improved doctor–patient relationships (DPRs). However, it is unclear how doctors and patients perceived the impact of doctors’ communication and empathy skills on DPRs during the COVID-19 pandemic. Objective To examine the perceptions of doctors and patients on how doctors’ communication skills and empathy skills influence DPRs during COVID-19. Main Measures Doctors’ and patients’ perceptions of doctors’ communication skills were measured using the Chinese version of the SEGUE Framework. To measure empathy skills and DPRs, the Jefferson Scale of Empathy and Difficult Doctor-Patient Relationship Questionnaire were administered to doctors, and the Consultation and Relational Empathy Measure and Patient-Doctor Relationship Questionnaire were administered to patients. Results A total of 902 doctors and 1432 patients in China were recruited during the pandemic via online or offline surveys (overall response rate of 69.8%). Both doctors and patients rated doctors’ empathy skills as more impactful on DPRs than communication skills. Doctors believed that only their empathy skills influenced DPRs. But patients believed that there was a significant bi-directional relationship between doctors’ communication and empathy skills and these two skills interacted to directly and indirectly influence DPRs, and doctors’ empathy had a greater mediating effect than their communication. Conclusions During COVID-19, there were both similarities and differences between Chinese doctors’ and patients’ views on how doctors’ communication and empathy skills influenced DPRs. The greater effect of doctors’ empathy skills suggests that both doctors and patients attach more importance to doctors’ empathy in doctor–patient interactions. The bi-directional effect on patient outcomes suggests that both doctors’ communication and empathy skills are important to patients’ perceptions of DPRs.

Determinants restricting ACE2 recognition of MERS-related coronaviruses in bats (preprint)

Phylogenetically distant coronaviruses have evolved to employ ACE2 as their common receptors, including NL63 and many Severe acute respiratory syndrome (SARS) coronavirus-related viruses. Recently, we found two Middle East respiratory syndrome coronaviruses (MERS-CoV)-related bat coronaviruses, NeoCoV and PDF-2180, also use Angiotensin-converting enzyme 2(ACE2) but not MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) for entry. Receptor binding domain (RBD)-binding and pseudovirus entry assays based on a wide range of bat ACE2 orthologs revealed that the two viruses strongly prefer ACE2 from Yangochiropteran bats as compared with Yinpterochiropteran bats, which is not observed in NL63 and SARS-CoV-2. Genetic and structural analyses of the virus-receptor interactions of 50 bat ACE2 orthologs pointed to four crucial host range determinants in two viral binding loops on ACE2. Subsequent functional verifications via mutagenesis on representative ACE2 orthologs confirmed the importance of these determinants on human and bat cells. Remarkably, NeoCoV-T510F, a mutation previously shown to acquire human ACE2 recognition, displayed an expanded potential host range covering most tested bat ACE2, probably due to its reinforced interaction with an evolutionary conserved hydrophobic pocket. Our results elucidated the molecular mechanisms for the species-specific ACE2 usage of MERS-related viruses, offering basic information for assessing the zoonotic risk of these ACE2 utilizing merbecoviruses.

The SARS‐CoV‐2 main protease (Mpro): Structure, function, and emerging therapies for COVID‐19

The main proteases (Mpro), also termed 3‐chymotrypsin‐like proteases (3CLpro), are a class of highly conserved cysteine hydrolases in β‐coronaviruses. Increasing evidence has demonstrated that 3CLpros play an indispensable role in viral replication and have been recognized as key targets for preventing and treating coronavirus‐caused infectious diseases, including COVID‐19. This review is focused on the structural features and biological function of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) main protease Mpro (also known as 3CLpro), as well as recent advances in discovering and developing SARS‐CoV‐2 3CLpro inhibitors. To better understand the characteristics of SARS‐CoV‐2 3CLpro inhibitors, the inhibition activities, inhibitory mechanisms, and key structural features of various 3CLpro inhibitors (including marketed drugs, peptidomimetic, and non‐peptidomimetic synthetic compounds, as well as natural compounds and their derivatives) are summarized comprehensively. Meanwhile, the challenges in this field are highlighted, while future directions for designing and developing efficacious 3CLpro inhibitors as novel anti‐coronavirus therapies are also proposed. Collectively, all information and knowledge presented here are very helpful for understanding the structural features and inhibitory mechanisms of SARS‐CoV‐2 3CLpro inhibitors, which offers new insights or inspiration to medicinal chemists for designing and developing more efficacious 3CLpro inhibitors as novel anti‐coronavirus agents. A comprehensive summary of recent advances in SARS‐CoV‐2 3CLpro inhibitors (including marketed drugs, peptidomimetic, and non‐peptidomimetic synthetic compounds, as well as natural compounds and their derivatives), including the inhibitory activities, inhibitory mechanisms, and key structural features, provides new insights for designing and developing more efficacious 3CLpro inhibitors as broad‐spectrum anti‐coronavirus agents.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors (preprint)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) and several bat coronaviruses employ Dipeptidyl peptidase-4 (DPP4) as their functional receptors. However, the receptor for NeoCoV, the closest MERS-CoV relative yet discovered in bats, remains enigmatic. In this study, we unexpectedly found that NeoCoV and its close relative, PDF-2180-CoV, can efficiently use some types of bat Angiotensin-converting enzyme 2 (ACE2) and, less favorably, human ACE2 for entry. The two viruses use their spikes' S1 subunit carboxyl-terminal domains (S1-CTD) for high-affinity and species-specific ACE2 binding. Cryo-electron microscopy analysis revealed a novel coronavirus-ACE2 binding interface and a protein-glycan interaction, distinct from other known ACE2-using viruses. We identified a molecular determinant close to the viral binding interface that restricts human ACE2 from supporting NeoCoV infection, especially around residue Asp338. Conversely, NeoCoV efficiently infects human ACE2 expressing cells after a T510F mutation on the receptor-binding motif (RBM). Notably, the infection could not be cross-neutralized by antibodies targeting SARS-CoV-2 or MERS-CoV. Our study demonstrates the first case of ACE2 usage in MERS-related viruses, shedding light on a potential bio-safety threat of the human emergence of an ACE2 using 'MERS-CoV-2' with both high fatality and transmission rate.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors (preprint)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) and several bat coronaviruses employ Dipeptidyl peptidase-4 (DPP4) as their functional receptors. However, the receptor for NeoCoV, the closest MERS-CoV relative yet discovered in bats, remains enigmatic. In this study, we unexpectedly found that NeoCoV and its close relative, PDF-2180-CoV, can efficiently use some types of bat Angiotensin-converting enzyme 2 (ACE2) and, less favorably, human ACE2 for entry. The two viruses use their spikes' S1 subunit carboxyl-terminal domains (S1-CTD) for high-affinity and species-specific ACE2 binding. Cryo-electron microscopy analysis revealed a novel coronavirus-ACE2 binding interface and a protein-glycan interaction, distinct from other known ACE2-using viruses. We identified a molecular determinant close to the viral binding interface that restricts human ACE2 from supporting NeoCoV infection, especially around residue Asp338. Conversely, NeoCoV efficiently infects human ACE2 expressing cells after a T510F mutation on the receptor-binding motif (RBM). Notably, the infection could not be cross-neutralized by antibodies targeting SARS-CoV-2 or MERS-CoV. Our study demonstrates the first case of ACE2 usage in MERS-related viruses, shedding light on a potential bio-safety threat of the human emergence of an ACE2 using "MERS-CoV-2" with both high fatality and transmission rate.

Antibody neutralization to SARS-CoV-2 and variants after one year in Wuhan (preprint)

Most COVID-19 patients can build effective humoral immunity against SARS-CoV-2 after recovery . However, it remains unknown how long the protection can maintain and how efficiently it can protect people from the reinfection of the emerging SARS-CoV-2 variants. Here we evaluated the sera from 248 COVID-19 convalescents around one year post-infection in Wuhan, the earliest epicenter of SARS-CoV-2. We demonstrated that the SARS-CoV-2 immunoglobulin G (IgG) maintains at a high level and potently neutralizes the infection of the original strain (WT) and the B.1.1.7 variant in most patients. However, they showed varying degrees of efficacy reduction against the other variants of concern (P.1, B.1.525, and especially B.1.351) in a patient-specific manner. Mutations in RBD including K417N, E484K, and E484Q/L452R (B.1.617) remarkably impair the neutralizing activity of the convalescents' sera. Encouragingly, we found that a small fraction of patients' sera showed broad neutralization potency to multiple variants and mutants, suggesting the existence of broadly neutralizing antibodies recognizing the epitopes beyond the mutation sites. Our results suggest that the SARS-CoV-2 vaccination effectiveness relies more on the timely re-administration of the epitope-updated vaccine than the durability of the neutralizing antibodies.

Single-cell immune profiling of matched disease and recovery phase blood of COVID-19 patients reveals anti-LAG3 based immune checkpoint therapy as a promising option (preprint)

We profiled the circulating immune cells in COVID-19 patients during the active disease and recovery phases via single cell RNA, TCR and BCR sequencing in order to elucidate the key anti-virus adaptive immune responses, and explore potential immunomodulatory therapeutic strategies. T cell competence plays a dominant role in anti-SARS-CoV-2 immunity. Clonally expanded CD4 T effector cells averted CD8 T cell exhaustion, and expansion of multiple CD8 T effector/memory clones was correlated with rapid virus clearance. In contrast, the patients lacking CD4 TEFF cell clonal expansion harbored exhausted CD8 TEFF cells co-expressing multiple co-inhibitory molecules. In addition, more than 90% of the exhausted cells expressed at least 2 and more than half of exhausted cells expressed at least 4 inhibitory molecules. Furthermore, co-expression of LAG3, Galectin-9, and SLAMF6 is the inhibitory molecule expression signature of exhausted T cell population in SARS-CoV-2 infection. Our findings indicate that LAG3-based immune checkpoint blockade is a promising strategy for treating COVID-19.