No Substrate Left behind-Mining of Shotgun Proteomics Datasets Rescues Evidence of Proteolysis by SARS-CoV-2 3CLpro Main Protease.

Proteolytic processing is the most ubiquitous post-translational modification and regulator of protein function. To identify protease substrates, and hence the function of proteases, terminomics workflows have been developed to enrich and detect proteolytically generated protein termini from mass spectrometry data. The mining of shotgun proteomics datasets for such 'neo'-termini, to increase the understanding of proteolytic processing, is an underutilized opportunity. However, to date, this approach has been hindered by the lack of software with sufficient speed to make searching for the relatively low numbers of protease-generated semi-tryptic peptides present in non-enriched samples viable. We reanalyzed published shotgun proteomics datasets for evidence of proteolytic processing in COVID-19 using the recently upgraded MSFragger/FragPipe software, which searches data with a speed that is an order of magnitude greater than many equivalent tools. The number of protein termini identified was higher than expected and constituted around half the number of termini detected by two different N-terminomics methods. We identified neo-N- and C-termini generated during SARS-CoV-2 infection that were indicative of proteolysis and were mediated by both viral and host proteases-a number of which had been recently validated by in vitro assays. Thus, re-analyzing existing shotgun proteomics data is a valuable adjunct for terminomics research that can be readily tapped (for example, in the next pandemic where data would be scarce) to increase the understanding of protease function and virus-host interactions, or other diverse biological processes.

Molecular Interaction of Nonsense-Mediated mRNA Decay with Viruses.

The virus-host interaction is dynamic and evolutionary. Viruses have to fight with hosts to establish successful infection. Eukaryotic hosts are equipped with multiple defenses against incoming viruses. One of the host antiviral defenses is the nonsense-mediated mRNA decay (NMD), an evolutionarily conserved mechanism for RNA quality control in eukaryotic cells. NMD ensures the accuracy of mRNA translation by removing the abnormal mRNAs harboring pre-matured stop codons. Many RNA viruses have a genome that contains internal stop codon(s) (iTC). Akin to the premature termination codon in aberrant RNA transcripts, the presence of iTC would activate NMD to degrade iTC-containing viral genomes. A couple of viruses have been reported to be sensitive to the NMD-mediated antiviral defense, while some viruses have evolved with specific cis-acting RNA features or trans-acting viral proteins to overcome or escape from NMD. Recently, increasing light has been shed on the NMD-virus interaction. This review summarizes the current scenario of NMD-mediated viral RNA degradation and classifies various molecular means by which viruses compromise the NMD-mediated antiviral defense for better infection in their hosts.

Type I interferon signaling in SARS-CoV-2 associated neurocognitive disorder (SAND): Mapping host-virus interactions to an etiopathogenesis.

Epidemiological, clinical, and radiological studies have provided insights into the phenomenology and biological basis of cognitive impairment in COVID-19 survivors. Furthermore, its association with biomarkers associated with neuroinflammation and neurodegeneration supports the notion that it is a distinct aspect of LongCOVID syndrome with specific underlying biology. Accounting for the latter, translational studies on SARS-CoV-2's interactions with its hosts have provided evidence on type I interferon dysregulation, which is seen in neuroinflammatory and neurodegenerative diseases. To date, studies attempting to describe this overlap have only described common mechanisms. In this manuscript, we attempt to propose a mechanistic model based on the host-virus interaction hypothesis. We discuss the molecular basis for a SARS-CoV-2-associated neurocognitive disorder (SAND) focusing on specific genes and pathways with potential mechanistic implications, several of which have been predicted by Vavougios and their research group. Furthermore, our hypothesis links translational evidence on interferon-responsive gene perturbations introduced by SARS-CoV-2 and known dysregulated pathways in dementia. Discussion emphasizes the crosstalk between central and peripheral immunity via danger-associated molecular patterns in inducing SAND's emergence in the absence of neuroinfection. Finally, we outline approaches to identifying targets that are both testable and druggable, and could serve in the design of future clinical and translational studies.

Insights on the evolution of Coronavirinae in general, and SARS-CoV-2 in particular, through innovative biocomputational resources.

The structural proteins of coronaviruses portray critical information to address issues of classification, assembly constraints, and evolutionary pathways involving host shifts. We compiled 173 complete protein sequences from isolates belonging to the four genera of the subfamily Coronavirinae. We calculate a single matrix of viral distance as a linear combination of protein distances. The minimum spanning tree (MST) connecting the individuals captures the structure of their similarities. The MST re-capitulates the known phylogeny of Coronovirinae. Hosts were mapped onto the MST and we found a non-trivial concordance between host phylogeny and viral proteomic distance. We also study the chimerism in our dataset through computational simulations. We found evidence that structural units coming from loosely related hosts hardly give rise to feasible chimeras in nature. This work offers a fresh way to analyze features of SARS-CoV-2 and related viruses.

Swine Enteric Coronavirus: Diverse Pathogen-Host Interactions.

Swine enteric coronavirus (SeCoV) causes acute gastroenteritis and high mortality in newborn piglets. Since the last century, porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) have swept farms all over the world and caused substantial economic losses. In recent years, porcine delta coronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV) have been emerging SeCoVs. Some of them even spread across species, which made the epidemic situation of SeCoV more complex and changeable. Recent studies have begun to reveal the complex SeCoV-host interaction mechanism in detail. This review summarizes the current advances in autophagy, apoptosis, and innate immunity induced by SeCoV infection. These complex interactions may be directly involved in viral replication or the alteration of some signal pathways.

Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites.

Glycans on the host cell membrane and viral proteins play critical roles in pathogenesis. Highly glycosylated epithelial cells represent the primary boundary separating embedded host tissues from pathogens within the respiratory and intestinal tracts. SARS-CoV-2, the causative agent for the COVID-19 pandemic, reaches into the respiratory tract. We found purified human milk oligosaccharides (HMOs) inhibited the viral binding on cells. Spike (S) protein receptor binding domain (RBD) binding to host cells were partly blocked by co-incubation with exogenous HMOs, most by 2-6-sialyl-lactose (6'SL), supporting the notion that HMOs can function as decoys in defense against SARS-Cov2. To investigate the effect of host cell glycocalyx on viral adherence, we metabolically modified and confirmed with glycomic methods the cell surface glycome to enrich specific N-glycan types including those containing sialic acids, fucose, mannose, and terminal galactose. Additionally, Immunofluorescence studies demonstrated that the S protein preferentially binds to terminal sialic acids with α-(2,6)-linkages. Furthermore, site-specific glycosylation of S protein RBD and its human receptor ACE2 were characterized using LC-MS/MS. We then performed molecular dynamics calculations on the interaction complex to further explore the interactive complex between ACE2 and the S protein. The results showed that hydrogen bonds mediated the interactions between ACE2 glycans and S protein with desialylated glycans forming significantly fewer hydrogen bonds. These results supported a mechanism where the virus binds initially to glycans on host cells preferring α-(2,6)-sialic acids and finds ACE2 and with the proper orientation infects the cell.

Innate Immunity Evasion Strategies of Highly Pathogenic Coronaviruses: SARS-CoV, MERS-CoV, and SARS-CoV-2.

In the past two decades, coronavirus (CoV) has emerged frequently in the population. Three CoVs (SARS-CoV, MERS-CoV, SARS-CoV-2) have been identified as highly pathogenic human coronaviruses (HP-hCoVs). Particularly, the ongoing COVID-19 pandemic caused by SARS-CoV-2 warns that HP-hCoVs present a high risk to human health. Like other viruses, HP-hCoVs interact with their host cells in sophisticated manners for infection and pathogenesis. Here, we reviewed the current knowledge about the interference of HP-hCoVs in multiple cellular processes and their impacts on viral infection. HP-hCoVs employed various strategies to suppress and evade from immune response, including shielding viral RNA from recognition by pattern recognition receptors (PRRs), impairing IFN-I production, blocking the downstream pathways of IFN-I, and other evasion strategies. This summary provides a comprehensive view of the interplay between HP-hCoVs and the host cells, which is helpful to understand the mechanism of viral pathogenesis and develop antiviral therapies.

Biology and Pathogenesis of SARS-CoV-2: Understandings for Therapeutic Developments against COVID-19.

Coronaviruses are positive sense, single-stranded, enveloped, and non-segmented RNA viruses that belong to the Coronaviridae family within the order Nidovirales and suborder Coronavirinae. Two Alphacoronavirus strains: HCoV-229E and HCoV-NL63 and five Betacoronaviruses: HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2 have so far been recognized as Human Coronaviruses (HCoVs). Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is currently the greatest concern for humanity. Despite the overflow of research on SARS-CoV-2 and other HCoVs published every week, existing knowledge in this area is insufficient for the complete understanding of the viruses and the diseases caused by them. This review is based on the analysis of 210 published works, and it attempts to cover the basic biology of coronaviruses, including the genetic characteristics, life cycle, and host-pathogen interaction, pathogenesis, the antiviral drugs, and vaccines against HCoVs, especially focusing on SARS-CoV-2. Furthermore, we will briefly discuss the potential link between extracellular vesicles (EVs) and SARS-CoV-2/COVID-19 pathophysiology.

Using Yeast to Identify Coronavirus-Host Protein Interactions.

We have developed a screening system using the yeast Saccharomyces cerevisiae to identify eukaryotic genes involved in the replication of mammalian viruses. Yeast come with various advantages, but in the context of coronavirus research and the system outlined here, they are simple and easy to work with and can be used at biosafety level 2. The system involves inducible expression of individual viral proteins and identification of detrimental phenotypes in the yeast. Yeast knockout and overexpression libraries can then be used for genome-wide screening of host proteins that provide a suppressor phenotype. From the yeast hits, a narrowed list of candidate genes can be produced to investigate for roles in viral replication. Since the system only requires expression of viral proteins, it can be used for any current or emerging virus, regardless of biocontainment requirements and ability to culture the virus. In this chapter, we will outline the protocols that can be used to take advantage of S. cerevisiae as a tool to advance understanding of how viruses interact with eukaryotic cells.

Repurposing of approved drugs with potential to interact with SARS-CoV-2 receptor.

Respiratory transmission is the primary route of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Angiotensin I converting enzyme 2 (ACE2) is the known receptor of SARS-CoV-2 surface spike glycoprotein for entry into human cells. A recent study reported absent to low expression of ACE2 in a variety of human lung epithelial cell samples. Three bioprojects (PRJEB4337, PRJNA270632 and PRJNA280600) invariably found abundant expression of ACE1 (a homolog of ACE2 and also known as ACE) in human lungs compared to very low expression of ACE2. In fact, ACE1 has a wider and more abundant tissue distribution compared to ACE2. Although it is not obvious from the primary sequence alignment of ACE1 and ACE2, comparison of X-ray crystallographic structures show striking similarities in the regions of the peptidase domains (PD) of these proteins, which is known (for ACE2) to interact with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Critical amino acids in ACE2 that mediate interaction with the viral spike protein are present and organized in the same order in the PD of ACE1. In silico analysis predicts comparable interaction of SARS-CoV-2 spike protein with ACE1 and ACE2. In addition, this study predicts from a list of 1263 already approved drugs that may interact with ACE2 and/or ACE1 and potentially interfere with the entry of SARS-CoV-2 inside the host cells.

An immune epigenetic insight to COVID-19 infection.

Severe acute respiratory syndrome coronavirus-2 is a positive-sense RNA virus, a causal agent of ongoing COVID-19 pandemic. ACE2R methylation across three CpG sites (cg04013915, cg08559914, cg03536816) determines the host cell's entry. It regulates ACE2 expression by controlling the SIRT1 and KDM5B activity. Further, it regulates Type I and III IFN response by modulating H3K27me3 and H3K4me3 histone mark. SARS-CoV-2 protein with bromodomain and protein E mimics bromodomain histones and evades from host immune response. The 2'-O MTases mimics the host's cap1 structure and plays a vital role in immune evasion through Hsp90-mediated epigenetic process to hijack the infected cells. Although the current review highlighted the critical epigenetic events associated with SARS-CoV-2 immune evasion, the detailed mechanism is yet to be elucidated.

The 20th Anniversary Meeting of the Rocky Mountain Virology Association.

Due to the COVID-19 pandemic and multiple devastating forest fires, the 2020 meeting of the Rocky Mountain Virology Association was held virtually. The three-day gathering featured talks describing recent advances in virology and prion research. The keynote presentation described the measles virus paradox of immune suppression and life-long immunity. Special invited speakers presented information concerning visualizing antiviral effector cell biology in mucosal tissues, uncovering the T-cell tropism of Epstein-Barr virus type 2, a history and current survey of coronavirus spike proteins, a summary of Zika virus vaccination and immunity, the innate immune response to flavivirus infections, a discussion concerning prion disease as it relates to multiple system atrophy, and clues for discussing virology with the non-virologist. On behalf of the Rocky Mountain Virology Association, this report summarizes selected presentations.

Autoimmunity as the comet tail of COVID-19 pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can give rise to different clinical manifestations that are directly related to viral tissue damage or indirectly induced by the antiviral immune response. Hyper-activation of the immune system in an attempt to eradicate the infection may trigger autoimmunity. Several immune-mediated disorders have been described in SARS-CoV-2-infected individuals. These include cutaneous rashes and vasculitis, autoimmune cytopenia, anti-phospholipid syndrome, central or peripheral neuropathy, myositis and myocarditis. On the other hand, rheumatic patients were reported to have similar coronavirus disease 2019 (COVID-19) incidence, morbidity and mortality rates compared to general population. This opinion review will summarize the crucial immunologic steps which occur during SARS-CoV-2-infection that may link autoimmunity to COVID-19 and provides an opportunity for further discussion regarding this association.

Leveraging on the genomics and immunopathology of SARS-CoV-2 for vaccines development: prospects and challenges.

The incidence and case-fatality rates (CFRs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, the etiological agent for Coronavirus Disease 2019 (COVID-19), have been rising unabated. Even though the entire world has been implementing infection prevention and control measures, the pandemic continues to spread. It has been widely accepted that preventive vaccination strategies are the public health measures for countering this pandemic. This study critically reviews the latest scientific advancement in genomics, replication pattern, pathogenesis, and immunopathology of SARS-CoV-2 infection and how these concepts could be used in the development of vaccines. We also offer a detailed discussion on the anticipated potency, efficacy, safety, and pharmaco-economic issues that are and will be associated with candidate COVID-19 vaccines.

SARS-CoV-2 multifaceted interaction with human host. Part I: What we have learnt and done so far, and the still unknown realities.

SARS-CoV-2, the causing agent of the ongoing COVID-19 pandemic, is a beta-coronavirus which has 80% genetic homology with SARS-CoV, but displays increased virulence and transmissibility. Initially, SARS-CoV-2 was considered a respiratory virus generally causing a mild disease, only severe and fatal in the elderly and individuals with underlying conditions. Severe illnesses and fatalities were attributed to a cytokine storm, an excessive response from the host immune system. However, with the number of infections over 10 millions and still soaring, the insidious and stealthy nature of the virus has emerged, as it causes a vast array of diverse unexpected symptoms among infected individuals, including the young and healthy. It has become evident that besides infecting the respiratory tract, SARS-CoV-2 can affect many organs, possibly through the infection of the endothelium. This review presents an overview of our learning curve with the novel virus emergence, transmission, pathology, biological properties and host-interactions. It also briefly describes remedial measures taken until an effective vaccine is available, that is non-pharmaceutical interventions to reduce the viral spread and the repurposing of existing drugs, approved or in development for other conditions to eliminate the virus or mitigate the cytokine storm.

Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression found in more than 200 diverse organisms. Although it is still not fully established if RNA viruses could generate miRNAs, there are examples of miRNA like sequences from RNA viruses with regulatory functions. In the case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with viral replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA-based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Overall, 950 hairpin structured sequences were extracted from the virus genome and based on the prediction results, 29 of them could be precursor miRNAs. Targeting analysis showed that 30 viral mature miRNA-like sequences could target 1,367 different human genes. PANTHER gene function analysis results indicated that viral derived miRNA candidates could target various human genes involved in crucial cellular processes including transcription, metabolism, defense system and several signaling pathways such as Wnt and EGFR signalings. Protein class-based grouping of targeted human genes showed that host transcription might be one of the main targets of the virus since 96 genes involved in transcriptional processes were potential targets of predicted viral miRNAs. For instance, basal transcription machinery elements including several components of human mediator complex (MED1, MED9, MED12L, MED19), basal transcription factors such as TAF4, TAF5, TAF7L and site-specific transcription factors such as STAT1 were found to be targeted. In addition, many known human miRNAs appeared to be able to target viral genes involved in viral life cycle such as S, M, N, E proteins and ORF1ab, ORF3a, ORF8, ORF7a and ORF10. Considering the fact that miRNA-based therapies have been paid attention, based on the findings of this study, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics.