The COVID-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified HCoV-19 virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 S protein, 20 are completely occupied by N -glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N -glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S and hACE2. Additional post-translational modifications (PTM) was identified, including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2. Refined structural models of HCoV-19 S and hACE2 were built by adding N -glycan and PTMs to recently published cryogenic electron microscopy (cryo-EM) structures. The PTM and glycan maps of HCoV-19 S and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19, as well as knowledge for developing desperately needed remedies and vaccines.

Enteroviruses (EVs) are major causes of viral meningoencephalitis in children. To better understand the pathogenesis and identify potential biomarkers, cerebrospinal fluid proteome in children (n = 52) suffering from EV meningoencephalitis was compared to that in EV-negative control subjects (n = 53) using the BoxCar acquisition technique. Among 1697 proteins identified, 1193 with robust assay readouts were used for quantitative analyses. Differential expression analyses identified 154 upregulated and 227 downregulated proteins in the EV-positive group. Functional analyses showed that the upregulated proteins are mainly related to activities of lymphocytes and cytokines, inflammation, and responses to stress and viral invasion, while the downregulated proteins are mainly related to neuronal integrity and activity as well as neurogenesis. According to receiver operating characteristic analysis results, Rho-GDP-dissociation inhibitor 2 exhibited the highest sensitivity (96.2%) and specificity (100%) for discriminating EV-positive from EV-negative patients. The chemokine CXCL10 was most upregulated (>300-fold) with also high sensitivity (92.3%) and specificity (94.3%) for indicating EV positivity. Thus, this study uncovered perturbations of multiple host processes due to EV meningoencephalitis, especially the general trend of enhanced immune responses but impaired neuronal functions. The identified dysregulated proteins may also prompt biomarker development.

The pneumonia-causing COVID-19 pandemia has prompt worldwide efforts to understand its biological and clinical traits of newly identified HCoV-19 virus. In this study, post-translational modification (PTM) of recombinant HCoV-19 S and hACE2 were characterized by LC-MSMS. We revealed that both proteins were highly decorated with specific proportions of N-glycan subtypes. Out of 21 possible glycosites in HCoV-19 S protein, 20 were confirmed completely occupied by N-glycans, with oligomannose glycans being the most abundant type. All 7 possible glycosylation sites in hACE2 were completely occupied mainly by complex type N-glycans. However, we showed that glycosylation did not directly contribute to the binding affinity between SARS-CoV spike protein and hACE2. Additionally, we also identified multiple sites methylated in both proteins, and multiple prolines in hACE2 were converted to hydroxylproline. Refined structural models were built by adding N-glycan and PTMs to recently published cryo-EM structure of the HCoV-19 S and hACE2 generated with glycosylation sites in the vicinity of binding surface. The PTM and glycan maps of both HCoV-19 S and hACE2 provide additional structural details to study mechanisms underlying host attachment, immune response mediated by S protein and hACE2, as well as knowledge to develop remedies and vaccines desperately needed nowadays.

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The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion. In addition to phosphoryl groups, we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs, including N -glycans and ubiquitin. Based on newly identified PTMs, refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs. These data can facilitate the design of novel vaccines or therapeutics targeting NCP, as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.