miRNAs, from Evolutionary Junk to Possible Prognostic Markers and Therapeutic Targets in COVID-19.

The COVID-19 pandemic has been a public health issue around the world in the last few years. Currently, there is no specific antiviral treatment to fight the disease. Thus, it is essential to highlight possible prognostic predictors that could identify patients with a high risk of developing complications. Within this framework, miRNA biomolecules play a vital role in the genetic regulation of various genes, principally, those related to the pathophysiology of the disease. Here, we review the interaction of host and viral microRNAs with molecular and cellular elements that could potentiate the main pulmonary, cardiac, renal, circulatory, and neuronal complications in COVID-19 patients. miR-26a, miR-29b, miR-21, miR-372, and miR-2392, among others, have been associated with exacerbation of the inflammatory process, increasing the risk of a cytokine storm. In addition, increased expression of miR-15b, -199a, and -491 are related to the prognosis of the disease, and miR-192 and miR-323a were identified as clinical predictors of mortality in patients admitted to the intensive care unit. Finally, we address miR-29, miR-122, miR-155, and miR-200, among others, as possible therapeutic targets. However, more studies are required to confirm these findings.

The Eukaryotic Linear Motif resource: 2022 release.

Almost twenty years after its initial release, the Eukaryotic Linear Motif (ELM) resource remains an invaluable source of information for the study of motif-mediated protein-protein interactions. ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at: http://elm.eu.org.

SARS-CoV-2 infection activates CREB/CBP in cellular cyclic AMP-dependent pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global coronavirus disease 2019 (COVID-19) pandemic that has affected the lives of billions of individuals. However, the host-virus interactions still need further investigation to reveal the underling mechanism of SARS-CoV-2 pathogenesis. Here, transcriptomics analysis of SARS-CoV-2 infection highlighted possible correlation between host-associated signaling pathway and virus. In detail, cAMP-protein kinase (PKA) pathway has an essential role in SARS-CoV-2 infection, followed by the interaction between cyclic AMP response element binding protein (CREB) and CREB-binding protein (CBP) could be induced and leading to the enhancement of CREB/CBP transcriptional activity. The replication of Delta and Omicron BA.5 were inhibited by about 49.4% and 44.7% after knockdown of CREB and CBP with small interfering RNAs, respectively. Furthermore, a small organic molecule naphthol AS-E (nAS-E), which targets on the interaction between CREB and CBP, potently inhibited SARS-CoV-2 wild-type (WT) infection with comparable the half-maximal effective concentration (EC50 ) 1.04 µM to Remdesivir 0.57 µM. Compared with WT virus, EC50 in Calu-3 cells against Delta, Omicron BA.2, and Omicron BA.5 were, on average, 1.5-fold, 1.1-fold, and 1.5-fold higher, respectively, nAS-E had a satisfied antiviral effect against Omicron variants. Taken together, our study demonstrated the importance of CREB/CBP induced by cAMP-PKA pathway during SARS-CoV-2 infection, and further provided a novel CREB/CBP interaction therapeutic drug targets for COVID-19.

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions.

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as regulators of various biological processes. Notably, emerging evidence demonstrates that host circRNAs can be differentially expressed (DE) upon infection with pathogens (e.g., influenza and coronaviruses), suggesting a role for circRNAs in regulating host innate immune responses. However, investigations on the role of circRNAs during pathogenic infections are limited by the knowledge and skills required to carry out the necessary bioinformatic analysis to identify DE circRNAs from RNA sequencing (RNA-seq) data. Bioinformatics prediction and identification of circRNAs is crucial before any verification, and functional studies using costly and time-consuming wet-lab techniques. To solve this issue, a step-by-step protocol of in silico prediction and characterization of circRNAs using RNA-seq data is provided in this manuscript. The protocol can be divided into four steps: 1) Prediction and quantification of DE circRNAs via the CIRIquant pipeline; 2) Annotation via circBase and characterization of DE circRNAs; 3) CircRNA-miRNA interaction prediction through Circr pipeline; 4) functional enrichment analysis of circRNA parental genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). This pipeline will be useful in driving future in vitro and in vivo research to further unravel the role of circRNAs in host-pathogen interactions.

Systematic comparison of published host gene expression signatures for bacterial/viral discrimination.

BACKGROUND: Measuring host gene expression is a promising diagnostic strategy to discriminate bacterial and viral infections. Multiple signatures of varying size, complexity, and target populations have been described. However, there is little information to indicate how the performance of various published signatures compare to one another. METHODS: This systematic comparison of host gene expression signatures evaluated the performance of 28 signatures, validating them in 4589 subjects from 51 publicly available datasets. Thirteen COVID-specific datasets with 1416 subjects were included in a separate analysis. Individual signature performance was evaluated using the area under the receiving operating characteristic curve (AUC) value. Overall signature performance was evaluated using median AUCs and accuracies. RESULTS: Signature performance varied widely, with median AUCs ranging from 0.55 to 0.96 for bacterial classification and 0.69-0.97 for viral classification. Signature size varied (1-398 genes), with smaller signatures generally performing more poorly (P < 0.04). Viral infection was easier to diagnose than bacterial infection (84% vs. 79% overall accuracy, respectively; P < .001). Host gene expression classifiers performed more poorly in some pediatric populations (3 months-1 year and 2-11 years) compared to the adult population for both bacterial infection (73% and 70% vs. 82%, respectively; P < .001) and viral infection (80% and 79% vs. 88%, respectively; P < .001). We did not observe classification differences based on illness severity as defined by ICU admission for bacterial or viral infections. The median AUC across all signatures for COVID-19 classification was 0.80 compared to 0.83 for viral classification in the same datasets. CONCLUSIONS: In this systematic comparison of 28 host gene expression signatures, we observed differences based on a signature's size and characteristics of the validation population, including age and infection type. However, populations used for signature discovery did not impact performance, underscoring the redundancy among many of these signatures. Furthermore, differential performance in specific populations may only be observable through this type of large-scale validation.

Flavivirus-host interactions: an expanding network of proviral and antiviral factors.

Flaviviruses are zoonotic pathogens transmitted by the bite of infected mosquitos and ticks and represent a constant burden to human health. Here we review recent literature aimed at uncovering how flaviviruses interact with the cells that they infect. A better understanding of these interactions may ultimately lead to novel therapeutic targets. We highlight several studies that employed low-biased methods to discover new protein-protein, protein-RNA, and genetic interactions, and spotlight recent work characterizing the host protein, TMEM41B, which has been shown to be critical for infection by diverse flaviviruses and coronaviruses.

Insights from a computational analysis of the SARS-CoV-2 Omicron variant: Host-pathogen interaction, pathogenicity, and possible drug therapeutics.

INTRODUCTION: Prominently accountable for the upsurge of COVID-19 cases as the world attempts to recover from the previous two waves, Omicron has further threatened the conventional therapeutic approaches. The lack of extensive research regarding Omicron has raised the need to establish correlations to understand this variant by structural comparisons. Here, we evaluate, correlate, and compare its genomic sequences through an immunoinformatic approach to understand its epidemiological characteristics and responses to existing drugs. METHODS: We reconstructed the phylogenetic tree and compared the mutational spectrum. We analyzed the mutations that occurred in the Omicron variant and correlated how these mutations affect infectivity and pathogenicity. Then, we studied how mutations in the receptor-binding domain affect its interaction with host factors through molecular docking. Finally, we evaluated the drug efficacy against the main protease of the Omicron through molecular docking and validated the docking results with molecular dynamics simulation. RESULTS: Phylogenetic and mutational analysis revealed the Omicron variant is similar to the highly infectious B.1.620 variant, while mutations within the prominent proteins are hypothesized to alter its pathogenicity. Moreover, docking evaluations revealed significant differences in binding affinity with human receptors, angiotensin-converting enzyme 2 and NRP1. Surprisingly, most of the tested drugs were proven to be effective. Nirmatrelvir, 13b, and Lopinavir displayed increased effectiveness against Omicron. CONCLUSION: Omicron variant may be originated from the highly infectious B.1.620 variant, while it was less pathogenic due to the mutations in the prominent proteins. Nirmatrelvir, 13b, and Lopinavir would be the most effective, compared to other promising drugs that were proven effective.

Bioinformatics-Based Approaches to Study Virus-Host Interactions During SARS-CoV-2 Infection.

As the knowledge of biomolecules is increasing from the last decades, it is helping the researchers to understand the unsolved issues regarding virology. Recent technologies in high-throughput sequencing are providing the swift generation of SARS-CoV-2 genomic data with the basic inside of viral infection. Owing to various virus-host protein interactions, high-throughput technologies are unable to provide complete details of viral pathogenesis. Identifying the virus-host protein interactions using bioinformatics approaches can assist in understanding the mechanism of SARS-CoV-2 infection and pathogenesis. In this chapter, recent integrative bioinformatics approaches are discussed to help the virologists and computational biologists in the identification of structurally similar proteins of human and SARS-CoV-2 virus, and to predict the potential of virus-host interactions. Considering experimental and time limitations for effective viral drug development, computational aided drug design (CADD) can reduce the gap between drug prediction and development. More research with respect to evolutionary solutions could be helpful to make a new pipeline for virus-host protein-protein interactions and provide more understanding to disclose the cases of host switch, and also expand the virulence of the pathogen and host range in developing viral infections.

A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection.

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1WT and HSP90AB1KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1WT and HSP90AB1KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1.

The roles of Eph receptors, neuropilin-1, P2X7, and CD147 in COVID-19-associated neurodegenerative diseases: inflammasome and JaK inhibitors as potential promising therapies.

The novel coronavirus disease 2019 (COVID-19) pandemic has spread worldwide, and finding a safe therapeutic strategy and effective vaccine is critical to overcoming severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, elucidation of pathogenesis mechanisms, especially entry routes of SARS-CoV-2 may help propose antiviral drugs and novel vaccines. Several receptors have been demonstrated for the interaction of spike (S) protein of SARS-CoV-2 with host cells, including angiotensin-converting enzyme (ACE2), ephrin ligands and Eph receptors, neuropilin 1 (NRP-1), P2X7, and CD147. The expression of these entry receptors in the central nervous system (CNS) may make the CNS prone to SARS-CoV-2 invasion, leading to neurodegenerative diseases. The present review provides potential pathological mechanisms of SARS-CoV-2 infection in the CNS, including entry receptors and cytokines involved in neuroinflammatory conditions. Moreover, it explains several neurodegenerative disorders associated with COVID-19. Finally, we suggest inflammasome and JaK inhibitors as potential therapeutic strategies for neurodegenerative diseases.

Whole-genome sequencing reveals host factors underlying critical COVID-19.

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2-4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease.

SARS-CoV-2 infections in children: Understanding diverse outcomes.

SARS-CoV-2 infections mostly lead to mild or even asymptomatic infections in children, but the reasons for this are not fully understood. More efficient local tissue responses, better thymic function, and cross-reactive immunity have all been proposed to explain this. In rare cases of children and young people, but very rarely in adults, post-infectious hyperinflammatory syndromes can develop and be serious. Here, I will discuss our current understanding of SARS-CoV-2 infections in children and hypothesize that a life history and energy allocation perspective might offer an additional explanation to mild infections, viral dynamics, and the higher incidence of rare multisystem inflammatory syndromes in children and young people.

Targeting stem-loop 1 of the SARS-CoV-2 5' UTR to suppress viral translation and Nsp1 evasion.

SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5' untranslated region (5' UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5' UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2-induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus' own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.

The low abundance of CpG in the SARS-CoV-2 genome is not an evolutionarily signature of ZAP.

The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.

Swine Enteric Coronaviruses (PEDV, TGEV, and PDCoV) Induce Divergent Interferon-Stimulated Gene Responses and Antigen Presentation in Porcine Intestinal Enteroids.

Swine enteric coronaviruses (SECoVs) including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV), account for the majority of lethal watery diarrhea in neonatal pigs and pose significant economic and public health burdens in the world. While the three SECoVs primarily infect intestinal epithelia in vivo and cause similar clinical signs, there are evident discrepancies in their cellular tropism and pathogenicity. However, the underlying mechanisms to cause the differences remain unclear. Herein, we employed porcine enteroids that are a physiologically relevant model of the intestine to assess the host epithelial responses following infection with the three SECoVs (PEDV, TGEV, and PDCoV). Although SECoVs replicated similarly in jejunal enteroids, a parallel comparison of transcriptomics datasets uncovered that PEDV and TGEV infection induced similar transcriptional profiles and exhibited a more pronounced response with more differentially expressed genes (DEGs) in jejunal enteroids compared with PDCoV infection. Notably, TGEV and PDCoV induced high levels of type I and III IFNs and IFN-stimulated gene (ISG) responses, while PEDV displayed a delayed peak and elicited a much lesser extent of IFN responses. Furthermore, TGEV and PDCoV instead of PEDV elicited a substantial upregulation of antigen-presentation genes and T cell-recruiting chemokines in enteroids. Mechanistically, we demonstrated that IFNs treatment markedly elevated the expression of NOD-like receptor (NLR) family NLRC5 and major histocompatibility complex class I (MHC-I) molecules. Together, our results indicate unique and common viral strategies for manipulating the global IFN responses and antigen presentation utilized by SECoVs, which help us a better understanding of host-SECoVs interactions.

Proteomic landscape of SARS-CoV-2- and MERS-CoV-infected primary human renal epithelial cells.

Acute kidney injury is associated with mortality in COVID-19 patients. However, host cell changes underlying infection of renal cells with SARS-CoV-2 remain unknown and prevent understanding of the molecular mechanisms that may contribute to renal pathology. Here, we carried out quantitative translatome and whole-cell proteomics analyses of primary renal proximal and distal tubular epithelial cells derived from human donors infected with SARS-CoV-2 or MERS-CoV to disseminate virus and cell type-specific changes over time. Our findings revealed shared pathways modified upon infection with both viruses, as well as SARS-CoV-2-specific host cell modulation driving key changes in innate immune activation and cellular protein quality control. Notably, MERS-CoV infection-induced specific changes in mitochondrial biology that were not observed in response to SARS-CoV-2 infection. Furthermore, we identified extensive modulation in pathways associated with kidney failure that changed in a virus- and cell type-specific manner. In summary, we provide an overview of the effects of SARS-CoV-2 or MERS-CoV infection on primary renal epithelial cells revealing key pathways that may be essential for viral replication.

Impact of ADAR-induced editing of minor viral RNA populations on replication and transmission of SARS-CoV-2.

Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (A→G) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in the early phase of the COVID-19 pandemic. A→G mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which A→G was more prevalent than any other mutation (P < 0.001). The A→G substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of A→G mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed A→G mutations in 288,247 SARS-CoV-2 major (consensus) sequences representing the dominant viral population. The A→G mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity.

SARS-CoV-2-Encoded MiRNAs Inhibit Host Type I Interferon Pathway and Mediate Allelic Differential Expression of Susceptible Gene.

Infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the rapid spread of coronavirus disease 2019 (COVID-19), has generated a public health crisis worldwide. The molecular mechanisms of SARS-CoV-2 infection and virus-host interactions are still unclear. In this study, we identified four unique microRNA-like small RNAs encoded by SARS-CoV-2. SCV2-miR-ORF1ab-1-3p and SCV2-miR-ORF1ab-2-5p play an important role in evasion of type I interferon response through targeting several genes in type I interferon signaling pathway. Particularly worth mentioning is that highly expressed SCV2-miR-ORF1ab-2-5p inhibits some key genes in the host innate immune response, such as IRF7, IRF9, STAT2, OAS1, and OAS2. SCV2-miR-ORF1ab-2-5p has also been found to mediate allelic differential expression of COVID-19-susceptible gene OAS1. In conclusion, these results suggest that SARS-CoV-2 uses its miRNAs to evade the type I interferon response and links the functional viral sequence to the susceptible genetic background of the host.