SARS-CoV-2 Mutations Lead to a Decrease in the Number of Tissue-Specific MicroRNA-Binding Regions in the Lung.

RNA interference in vertebrates acts as an antiviral mechanism only in undifferentiated embryonic stem cells and is mediated by microRNAs. In somatic cells, host microRNAs also bind to the genomes of RNA viruses, regulating their translation and replication. It has been shown that viral (+)RNA can evolve under the influence of host cell miRNAs. In more than two years of the pandemic, the SARS-CoV-2 virus has mutated significantly. It is quite possible that some mutations could be retained in the virus genome under the influence of miRNAs produced by alveolar cells. We demonstrated that microRNAs in human lung tissue exert evolutionary pressure on the SARS-CoV-2 genome. Moreover, a significant number of sites of host microRNA binding with the virus genome are located in the NSP3-NSP5 region responsible for autoproteolysis of viral polypeptides.

MVIP: multi-omics portal of viral infection.

Virus infections are huge threats to living organisms and cause many diseases, such as COVID-19 caused by SARS-CoV-2, which has led to millions of deaths. To develop effective strategies to control viral infection, we need to understand its molecular events in host cells. Virus related functional genomic datasets are growing rapidly, however, an integrative platform for systematically investigating host responses to viruses is missing. Here, we developed a user-friendly multi-omics portal of viral infection named as MVIP (https://mvip.whu.edu.cn/). We manually collected available high-throughput sequencing data under viral infection, and unified their detailed metadata including virus, host species, infection time, assay, and target, etc. We processed multi-layered omics data of more than 4900 viral infected samples from 77 viruses and 33 host species with standard pipelines, including RNA-seq, ChIP-seq, and CLIP-seq, etc. In addition, we integrated these genome-wide signals into customized genome browsers, and developed multiple dynamic charts to exhibit the information, such as time-course dynamic and differential gene expression profiles, alternative splicing changes and enriched GO/KEGG terms. Furthermore, we implemented several tools for efficiently mining the virus-host interactions by virus, host and genes. MVIP would help users to retrieve large-scale functional information and promote the understanding of virus-host interactions.

Viral Circular RNAs and Their Possible Roles in Virus-Host Interaction.

Circular RNAs (circRNAs) as novel regulatory molecules have been recognized in diverse species, including viruses. The virus-derived circRNAs play various roles in the host biological process and the life cycle of the viruses. This review summarized the circRNAs from the DNA and RNA viruses and discussed the biogenesis of viral and host circRNAs, the potential roles of viral circRNAs, and their future perspective. This review will elaborate on new insights gained on viruses encoded circRNAs during virus infection.

DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

Rapid SARS-CoV-2 Intra-Host and Within-Household Emergence of Novel Haplotypes.

In February 2020, the municipality of Vo', a small town near Padua (Italy) was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. To investigate the viral prevalence and clinical features, the entire population was swab tested in two sequential surveys. Here we report the analysis of 87 viral genomes, which revealed that the unique ancestor haplotype introduced in Vo' belongs to lineage B, carrying the mutations G11083T and G26144T. The viral sequences allowed us to investigate the viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo'. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptom severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype that was carried by an index case. The lockdown extinguished both the viral spread and the emergence of new variants.

Computational Analysis of Mutations in the Receptor-Binding Domain of SARS-CoV-2 Spike and Their Effects on Antibody Binding.

Currently, SARS-CoV-2 causing coronavirus disease 2019 (COVID-19) is responsible for one of the most deleterious pandemics of our time. The interaction between the ACE2 receptors at the surface of human cells and the viral Spike (S) protein triggers the infection, making the receptor-binding domain (RBD) of the SARS-CoV-2 S-protein a focal target for the neutralizing antibodies (Abs). Despite the recent progress in the development and deployment of vaccines, the emergence of novel variants of SARS-CoV-2 insensitive to Abs produced in response to the vaccine administration and/or monoclonal ones represent a potential danger. Here, we analyzed the diversity of neutralizing Ab epitopes and assessed the possible effects of single and multiple mutations in the RBD of SARS-CoV-2 S-protein on its binding affinity to various antibodies and the human ACE2 receptor using bioinformatics approaches. The RBD-Ab complexes with experimentally resolved structures were grouped into four clusters with distinct features at sequence and structure level. The performed computational analysis indicates that while single amino acid replacements in RBD may only cause partial impairment of the Abs binding, moreover, limited to specific epitopes, the variants of SARS-CoV-2 with multiple mutations, including some which were already detected in the population, may potentially result in a much broader antigenic escape. Further analysis of the existing RBD variants pointed to the trade-off between ACE2 binding and antigenic escape as a key limiting factor for the emergence of novel SAR-CoV-2 strains, as the naturally occurring mutations in RBD tend to reduce its binding affinity to Abs but not to ACE2. The results provide guidelines for further experimental studies aiming to identify high-risk RBD mutations that allow for an antigenic escape.

Influenza-induced Tpl2 expression within alveolar epithelial cells is dispensable for host viral control and anti-viral immunity.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that regulates the expression of inflammatory mediators in response to Toll-like receptors (TLR) and cytokine receptors. Global ablation of Tpl2 leads to severe disease in response to influenza A virus (IAV) infection, characterized by respiratory distress, and studies in bone marrow chimeric mice implicated Tpl2 in non-hematopoietic cells. Lung epithelial cells are primary targets and replicative niches of influenza viruses; however, the specific regulation of antiviral responses by Tpl2 within lung epithelial cells has not been investigated. Herein, we show that Tpl2 is basally expressed in primary airway epithelial cells and that its expression increases in both type I and type II airway epithelial cells (AECI and AECII) in response to influenza infection. We used Nkx2.1-cre to drive Tpl2 deletion within pulmonary epithelial cells to delineate epithelial cell-specific functions of Tpl2 during influenza infection in mice. Although modest increases in morbidity and mortality were attributed to cre-dependent deletion in lung epithelial cells, no alterations in host cytokine production or lung pathology were observed. In vitro, Tpl2 inhibition within the type I airway epithelial cell line, LET1, as well as genetic ablation in primary airway epithelial cells did not alter cytokine production. Overall, these findings establish that Tpl2-dependent defects in cells other than AECs are primarily responsible for the morbidity and mortality seen in influenza-infected mice with global Tpl2 ablation.

Scanning the RBD-ACE2 molecular interactions in Omicron variant.

The emergence of new SARS-CoV-2 variants poses a threat to the human population where it is difficult to assess the severity of a particular variant of the virus. Spike protein and specifically its receptor binding domain (RBD) which makes direct interaction with the ACE2 receptor of the human has shown prominent amino acid substitutions in most of the Variants of Concern. Here, by using all-atom molecular dynamics simulations we compare the interaction of Wild-type RBD/ACE2 receptor complex with that of the latest Omicron variant of the virus. We observed a very interesting diversification of the charge, dynamics and energetics of the protein complex formed upon mutations. These results would help us in understanding the molecular basis of binding of the Omicron variant with that of SARS-CoV-2 Wild-type.

Structural modeling of Omicron spike protein and its complex with human ACE-2 receptor: Molecular basis for high transmissibility of the virus.

Omicron is a new variant of SARS-CoV-2, which is currently infecting people around the world. Spike glycoprotein, an important molecule in pathogenesis of infection has been modeled and the interaction of its Receptor Binding Domain with human ACE-receptor has been analysed by simulation studies. Structural analysis of Omicron spike glycoprotein shows the 30 mutations to be distributed over all domains of the trimeric protein, wherein the mutant residues are seen to be participating in higher number of intra-molecular interactions including two salt bridges emanating from mutant residues thereby stabilizing their conformation, as compared to wild type. Complex of Receptor Binding Domain (RBD) with human ACE-2 receptor shows seven mutations at interacting interface comprising of two ionic interactions, eight hydrogen bonds and seven Van der Waals interactions. The number and quality of these interactions along with other binding biophysical parameters suggests more potency of RBD domain to the receptor as compared to the wild type counterpart. Results of this study explains the high transmissibility of Omicron variant of SARS-CoV-2 that is currently observed across the world.

Viral surface geometry shapes influenza and coronavirus spike evolution through antibody pressure.

The evolution of circulating viruses is shaped by their need to evade antibody response, which mainly targets the viral spike. Because of the high density of spikes on the viral surface, not all antigenic sites are targeted equally by antibodies. We offer here a geometry-based approach to predict and rank the probability of surface residues of SARS spike (S protein) and influenza H1N1 spike (hemagglutinin) to acquire antibody-escaping mutations utilizing in-silico models of viral structure. We used coarse-grained MD simulations to estimate the on-rate (targeting) of an antibody model to surface residues of the spike protein. Analyzing publicly available sequences, we found that spike surface sequence diversity of the pre-pandemic seasonal influenza H1N1 and the sarbecovirus subgenus highly correlates with our model prediction of antibody targeting. In particular, we identified an antibody-targeting gradient, which matches a mutability gradient along the main axis of the spike. This identifies the role of viral surface geometry in shaping the evolution of circulating viruses. For the 2009 H1N1 and SARS-CoV-2 pandemics, a mutability gradient along the main axis of the spike was not observed. Our model further allowed us to identify key residues of the SARS-CoV-2 spike at which antibody escape mutations have now occurred. Therefore, it can inform of the likely functional role of observed mutations and predict at which residues antibody-escaping mutation might arise.

Molecular dynamic simulation analysis of SARS-CoV-2 spike mutations and evaluation of ACE2 from pets and wild animals for infection risk.

Coronavirus Disease 2019 (COVID-19) is an ongoing global health emergency that has caused tremendous stress and loss of life worldwide. The viral spike glycoprotein is a critical molecule mediating transmission of SARS-CoV-2 by interacting with human ACE2. However, through the course of the pandemics, there has not been a thorough analysis of the spike protein mutations, and on how these mutants influence the transmission of SARS-CoV-2. Besides, cases of SARS-CoV-2 infection among pets and wild animals have been reported, so the susceptibility of these animals requires great attention to investigate, as they may also link to the renewed question of a possible intermediate host for SARS-CoV-2 before it was transmitted to humans. With over 226,000 SARS-CoV-2 sequences obtained, we found 1573 missense mutations in the spike gene, and 226 of them were within the receptor-binding domain (RBD) region that directly interacts with human ACE2. Modeling the interactions between SARS-CoV-2 spike mutants and ACE2 molecules showed that most of the 74 missense mutations in the RBD region of the interaction interface had little impact on spike binding to ACE2, whereas several within the spike RBD increased the binding affinity toward human ACE2 thus making the virus likely more contagious. On the other hand, modeling the interactions between animal ACE2 molecules and SARS-CoV-2 spike revealed that many pets and wild animals' ACE2 had a variable binding ability. Particularly, ACE2 of bamboo rat had stronger binding to SARS-CoV-2 spike protein, whereas that of mole, vole, Mus pahari, palm civet, and pangolin had a weaker binding compared to human ACE2. Our results provide structural insights into the impact on interactions of the SARS-CoV-2 spike mutants to human ACE2, and shed light on SARS-CoV-2 transmission in pets and wild animals, and possible clues to the intermediate host(s) for SARS-CoV-2.

Lung disease network reveals impact of comorbidity on SARS-CoV-2 infection and opportunities of drug repurposing.

BACKGROUND: Higher mortality of COVID-19 patients with lung disease is a formidable challenge for the health care system. Genetic association between COVID-19 and various lung disorders must be understood to comprehend the molecular basis of comorbidity and accelerate drug development. METHODS: Lungs tissue-specific neighborhood network of human targets of SARS-CoV-2 was constructed. This network was integrated with lung diseases to build a disease-gene and disease-disease association network. Network-based toolset was used to identify the overlapping disease modules and drug targets. The functional protein modules were identified using community detection algorithms and biological processes, and pathway enrichment analysis. RESULTS: In total, 141 lung diseases were linked to a neighborhood network of SARS-CoV-2 targets, and 59 lung diseases were found to be topologically overlapped with the COVID-19 module. Topological overlap with various lung disorders allows repurposing of drugs used for these disorders to hit the closely associated COVID-19 module. Further analysis showed that functional protein-protein interaction modules in the lungs, substantially hijacked by SARS-CoV-2, are connected to several lung disorders. FDA-approved targets in the hijacked protein modules were identified and that can be hit by exiting drugs to rescue these modules from virus possession. CONCLUSION: Lung diseases are clustered with COVID-19 in the same network vicinity, indicating the potential threat for patients with respiratory diseases after SARS-CoV-2 infection. Pathobiological similarities between lung diseases and COVID-19 and clinical evidence suggest that shared molecular features are the probable reason for comorbidity. Network-based drug repurposing approaches can be applied to improve the clinical conditions of COVID-19 patients.

Poking COVID-19: Insights on Genomic Constraints among Immune-Related Genes between Qatari and Italian Populations.

Host genomic information, specifically genomic variations, may characterize susceptibility to disease and identify people with a higher risk of harm, leading to better targeting of care and vaccination. Italy was the epicentre for the spread of COVID-19 in Europe, the first country to go into a national lockdown and has one of the highest COVID-19 associated mortality rates. Qatar, on the other hand has a very low mortality rate. In this study, we compared whole-genome sequencing data of 14398 adults and Qatari-national to 925 Italian individuals. We also included in the comparison whole-exome sequence data from 189 Italian laboratory-confirmed COVID-19 cases. We focused our study on a curated list of 3619 candidate genes involved in innate immunity and host-pathogen interaction. Two population-gene metric scores, the Delta Singleton-Cohort variant score (DSC) and Sum Singleton-Cohort variant score (SSC), were applied to estimate the presence of selective constraints in the Qatari population and in the Italian cohorts. Results based on DSC and SSC metrics demonstrated a different selective pressure on three genes (MUC5AC, ABCA7, FLNA) between Qatari and Italian populations. This study highlighted the genetic differences between Qatari and Italian populations and identified a subset of genes involved in innate immunity and host-pathogen interaction.

Emerging mutation patterns in SARS-CoV-2 variants.

There is an urgent need to understand the functional effects of mutations in emerging variants of SARS-CoV-2. Variants of concern (alpha, beta, gamma and delta) acquired four patterns of spike glycoprotein mutations that enhance transmissibility and immune evasion: 1) mutations in the N-terminal domain (NTD), 2) mutations in the Receptor Binding Domain (RBD), 3) mutations at interchain contacts of the spike trimer, and 4) furin cleavage site mutations. Most distinguishing mutations among variants of concern are exhibited in the NTD, localized to sites of high structural flexibility. Emerging variants of interest such as mu, lambda and C.1.2 exhibit the same patterns of mutations as variants of concern. There is a strong likelihood that SARS-CoV-2 variants will continue to emerge with mutations in these defined patterns, thus providing a basis for the development of next line antiviral drugs and vaccine candidates.

Comparative genomic analysis reveals varying levels of mammalian adaptation to coronavirus infections.

Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is of zoonotic origin. Evolutionary analyses assessing whether coronaviruses similar to SARS-CoV-2 infected ancestral species of modern-day animal hosts could be useful in identifying additional reservoirs of potentially dangerous coronaviruses. We reasoned that if a clade of species has been repeatedly exposed to a virus, then their proteins relevant for viral entry may exhibit adaptations that affect host susceptibility or response. We perform comparative analyses across the mammalian phylogeny of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, in order to uncover evidence for selection acting at its binding interface with the SARS-CoV-2 spike protein. We uncover that in rodents there is evidence for adaptive amino acid substitutions at positions comprising the ACE2-spike interaction interface, whereas the variation within ACE2 proteins in primates and some other mammalian clades is not consistent with evolutionary adaptations. We also analyze aminopeptidase N (APN), the receptor for the human coronavirus 229E, a virus that causes the common cold, and find evidence for adaptation in primates. Altogether, our results suggest that the rodent and primate lineages may have had ancient exposures to viruses similar to SARS-CoV-2 and HCoV-229E, respectively.

A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells.

The positive-sense, single-stranded RNA genome SARS-CoV-2 harbors functionally important cis-acting elements governing critical aspects of viral gene expression. However, insights on how these elements sense various signals from the host cell and regulate viral protein synthesis are lacking. Here, we identified two novel cis-regulatory elements in SARS-CoV-2 ORF1a and S RNAs and describe their role in translational control of SARS-CoV-2. These elements are sequence-unrelated but form conserved hairpin structures (validated by NMR) resembling gamma activated inhibitor of translation (GAIT) elements that are found in a cohort of human mRNAs directing translational suppression in myeloid cells in response to IFN-γ. Our studies show that treatment of human lung cells with receptor-binding S1 subunit, S protein pseudotyped lentivirus, and S protein-containing virus-like particles triggers a signaling pathway involving DAP-kinase1 that leads to phosphorylation and release of the ribosomal protein L13a from the large ribosomal subunit. Released L13a forms a virus activated inhibitor of translation (VAIT) complex that binds to ORF1a and S VAIT elements, causing translational silencing. Translational silencing requires extracellular S protein (and its interaction with host ACE2 receptor), but not its intracellular synthesis. RNA-protein interaction analyses and in vitro translation experiments showed that GAIT and VAIT elements do not compete with each other, highlighting differences between the two pathways. Sequence alignments of SARS-CoV-2 genomes showed a high level of conservation of VAIT elements, suggesting their functional importance. This VAIT-mediated translational control mechanism of SARS-CoV-2 may provide novel targets for small molecule intervention and/or facilitate development of more effective mRNA vaccines. IMPORTANCE Specific RNA elements in the genomes of RNA viruses play important roles in host-virus interaction. For SARS-CoV-2, the mechanistic insights on how these RNA elements could sense the signals from the host cell are lacking. Here we report a novel relationship between the GAIT-like SARS-CoV-2 RNA element (called VAITs) and the signal generated from the host cell. We show that for SARS-CoV-2, the interaction of spike protein with ACE2 not only serves the purpose for viral entry into the host cell, but also transduces signals that culminate into the phosphorylation and the release of L13a from the large ribosomal subunit. We also show that this event leads to the translational arrest of ORF1a and S mRNAs in a manner dependent on the structure of the RNA elements. Translational control of viral mRNA by a host-cell generated signal triggered by viral protein is a new paradigm in the host-virus relationship.

SARS-CoV-2 infection mediates differential expression of human endogenous retroviruses and long interspersed nuclear elements.

SARS-CoV-2 promotes an imbalanced host response that underlies the development and severity of COVID-19. Infections with viruses are known to modulate transposable elements (TEs), which can exert downstream effects by modulating host gene expression, innate immune sensing, or activities encoded by their protein products. We investigated the impact of SARS-CoV-2 infection on TE expression using RNA-Seq data from cell lines and from primary patient samples. Using a bioinformatics tool, Telescope, we showed that SARS-CoV-2 infection led to upregulation or downregulation of TE transcripts, a subset of which differed from cells infected with SARS, Middle East respiratory syndrome coronavirus (MERS-CoV or MERS), influenza A virus (IAV), respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (HPIV3). Differential expression of key retroelements specifically identified distinct virus families, such as Coronaviridae, with unique retroelement expression subdividing viral species. Analysis of ChIP-Seq data showed that TEs differentially expressed in SARS-CoV-2 infection were enriched for binding sites for transcription factors involved in immune responses and for pioneer transcription factors. In samples from patients with COVID-19, there was significant TE overexpression in bronchoalveolar lavage fluid and downregulation in PBMCs. Thus, although the host gene transcriptome is altered by infection with SARS-CoV-2, the retrotranscriptome may contain the most distinctive features of the cellular response to SARS-CoV-2 infection.

Genome Editing Strategies to Protect Livestock from Viral Infections.

The livestock industry is constantly threatened by viral disease outbreaks, including infections with zoonotic potential. While preventive vaccination is frequently applied, disease control and eradication also depend on strict biosecurity measures. Clustered regularly interspaced palindromic repeats (CRISPR) and associated proteins (Cas) have been repurposed as genome editors to induce targeted double-strand breaks at almost any location in the genome. Thus, CRISPR/Cas genome editors can also be utilized to generate disease-resistant or resilient livestock, develop vaccines, and further understand virus-host interactions. Genes of interest in animals and viruses can be targeted to understand their functions during infection. Furthermore, transgenic animals expressing CRISPR/Cas can be generated to target the viral genome upon infection. Genetically modified livestock can thereby reduce disease outbreaks and decrease zoonotic threats.

COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms.

We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective.

Epigenetic regulation in antiviral innate immunity.

Emerging life-threatening viruses have posed great challenges to public health. It is now increasingly clear that epigenetics plays a role in shaping host-virus interactions and there is a great need for a more thorough understanding of these intricate interactions through the epigenetic lens, which may represent potential therapeutic opportunities in the clinic. In this review, we highlight the current understanding of the roles of key epigenetic regulators - chromatin remodeling and histone modification - in modulating chromatin openness during host defense against virus. We also discuss how the RNA modification m6A (N6-methyladenosine) affects fundamental aspects of host-virus interactions. We conclude with future directions for uncovering more detailed functions that epigenetic regulation exerts on both host cells and viruses during infection.