Glycine 29 Is Critical for Conformational Changes of the Spike Glycoprotein of Mouse Hepatitis Virus A59 Triggered by either Receptor Binding or High pH.

Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696-10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis and identified I22 as being essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH. Replacement of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from the S protein, resulting in an enhancement of high-pH-triggered receptor-independent syncytium (RIS) formation in HEK293T cells, compared to the wild type (WT). In contrast, replacement of G29 with P, a turn-prone residue with a strict conformation, hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain (CTD) of the S1 subunit, indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered.IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the ß1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion.

Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59.

The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness.IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.

COVID-19 Antibody Levels among Various Vaccination Groups, One-Year Antibody Follow-Up in Two University Hospitals from Western and Central Turkey.

Various clinical outcomes, reinfections, vaccination programs, and antibody responses resulted from the COVID-19 pandemic. This study investigated the time-dependent changes in SARS-CoV-2 antibody responses in infected and/or vaccinated and unvaccinated individuals and to provide insights into spike and nucleocapsid antibodies, which fluctuate during infectious and non-infectious states. This cohort study was carried out at the Ege University Faculty of Medicine hospital in Izmir (western Turkey) and the Erciyes University Faculty of Medicine hospital in Kayseri (central Turkey) between December 2021 and January 2023, which coincided with the second half of COVID-19 pandemic. The study included 100 COVID-19 PCR-positive patients and 190 healthcare workers (HCWs). Antibody levels were followed up via quantitative anti-SARS-CoV-2 spike and qualitative anti-nucleocapsid immunoassays (Elecsys™). Antibody levels declined after infection but persisted for at least 6-8 months. Individuals who had received only CoronaVac had higher anti-nucleocapsid antibody levels in the early months than those who received mixed vaccination. However, anti-spike antibodies persisted longer and at higher levels in individuals who had received mixed vaccinations. This suggests that combining two different vaccine platforms may provide a synergistic effect, resulting in more durable and broad-spectrum immunity against SARS-CoV-2. The study provides information about the vaccination and antibody status of healthcare workers in the second half of the pandemic and provides valuable insights into the dynamics of antibody responses to COVID-19 infection and vaccination.

Genetic diversity and evolutionary patterns of SARS-CoV-2 among the Bhutanese population during the pandemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic, caused by a dynamic virus, has had a profound global impact. Despite declining global COVID-19 cases and mortality rates, the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains a major concern. This study provides a comprehensive analysis of the genomic sequences of SARS-CoV-2 within the Bhutanese population during the pandemic. The primary aim was to elucidate the molecular epidemiology and evolutionary patterns of SARS-CoV-2 in Bhutan, with a particular focus on genetic variations and lineage dynamics. METHODS: Whole-genome sequences of SARS-CoV-2 collected from Bhutan between May 2020 and February 2023 (n=135) were retrieved from the Global Initiative on Sharing All Influenza Database. RESULTS: The SARS-CoV-2 variants in Bhutan were predominantly classified within the Nextstrain clade 20A (31.1%), followed by clade 21L (20%) and clade 22D (15.6%). We identified 26 Pangolin lineages with variations in their spatial and temporal distribution. Bayesian time-scaled phylogenetic analysis estimated the time to the most recent common ancestor as February 15, 2020, with a substitution rate of 0.97×10-3 substitutions per site per year. Notably, the spike glycoprotein displayed the highest mutation frequency among major viral proteins, with 116 distinct mutations, including D614G. The Bhutanese isolates also featured mutations such as E484K, K417N, and S477N in the spike protein, which have implications for altered viral properties. CONCLUSION: This is the first study to describe the genetic diversity of SARS-CoV-2 circulating in Bhutan during the pandemic, and this data can inform public health policies and strategies for preventing future outbreaks in Bhutan.

Kathryn V. Holmes: A Career of Contributions to the Coronavirus Field.

Over the past two years, scientific research has moved at an unprecedented rate in response to the COVID-19 pandemic. The rapid development of effective vaccines and therapeutics would not have been possible without extensive background knowledge on coronaviruses developed over decades by researchers, including Kathryn (Kay) Holmes. Kay's research team discovered the first coronavirus receptors for mouse hepatitis virus and human coronavirus 229E and contributed a wealth of information on coronaviral spike glycoproteins and receptor interactions that are critical determinants of host and tissue specificity. She collaborated with several research laboratories to contribute knowledge in additional areas, including coronaviral pathogenesis, epidemiology, and evolution. Throughout her career, Kay was an extremely dedicated and thoughtful mentor to numerous graduate students and post-doctoral fellows. This article provides a review of her contributions to the coronavirus field and her exemplary mentoring.

SARS-CoV-2 E484K Mutation Narrative Review: Epidemiology, Immune Escape, Clinical Implications, and Future Considerations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly over the world and claimed million lives. The virus evolves constantly, and a swarm of mutants is a now major concern globally. Distinct variants could have independently converged on same mutation, despite being detected in different geographic regions, which suggested it could confer an evolutionary advantage. E484K has rapidly emerged and has frequently been detected in several SARS-CoV-2 variants of concern. In this study, we review the epidemiology and impact of E484K, its effects on neutralizing effect of several monoclonal antibodies, convalescent plasma, and post-vaccine sera.

Middle East Respiratory Syndrome Vaccine Candidates: Cautious Optimism.

Efforts towards developing a vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV) have yielded promising results. Utilizing a variety of platforms, several vaccine approaches have shown efficacy in animal models and begun to enter clinical trials. In this review, we summarize the current progress towards a MERS-CoV vaccine and highlight potential roadblocks identified from previous attempts to generate coronavirus vaccines.