Transcription Factor Driven Gene Regulation in COVID-19 Patients.

SARS-CoV-2 and its many variants have caused a worldwide emergency. Host cells colonised by SARS-CoV-2 present a significantly different gene expression landscape. As expected, this is particularly true for genes that directly interact with virus proteins. Thus, understanding the role that transcription factors can play in driving differential regulation in patients affected by COVID-19 is a focal point to unveil virus infection. In this regard, we have identified 19 transcription factors which are predicted to target human proteins interacting with Spike glycoprotein of SARS-CoV-2. Transcriptomics RNA-Seq data derived from 13 human organs are used to analyse expression correlation between identified transcription factors and related target genes in both COVID-19 patients and healthy individuals. This resulted in the identification of transcription factors showing the most relevant impact in terms of most evident differential correlation between COVID-19 patients and healthy individuals. This analysis has also identified five organs such as the blood, heart, lung, nasopharynx and respiratory tract in which a major effect of differential regulation mediated by transcription factors is observed. These organs are also known to be affected by COVID-19, thereby providing consistency to our analysis. Furthermore, 31 key human genes differentially regulated by the transcription factors in the five organs are identified and the corresponding KEGG pathways and GO enrichment are also reported. Finally, the drugs targeting those 31 genes are also put forth. This in silico study explores the effects of transcription factors on human genes interacting with Spike glycoprotein of SARS-CoV-2 and intends to provide new insights to inhibit the virus infection.

Replicating-Competent VSV-Vectored Pseudotyped Viruses.

Vesicular stomatitis virus (VSV) is prototype virus in the family of Rhabdoviridae. Reverse genetic platform has enabled the genetic manipulation of VSV as a powerful live viral vector. Replicating-competent VSV is constructed by replacing the original VSV glycoprotein gene with heterologous envelope genes. The resulting recombinant viruses are able to replicate in permissive cells and incorporate the foreign envelope proteins on the surface of the viral particle without changing the bullet-shape morphology. Correspondingly, the cell tropism of replicating-competent VSV is determined by the foreign envelope proteins. Replicating-competent VSVs have been successfully used for selecting critical viral receptors or host factors, screening mutants that escape therapeutic antibodies, and developing VSV-based live viral vaccines.

Genotyping and In Silico Analysis of Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) Spike 1 (S1) Glycoprotein.

Genetic diversity and evolution of infectious bronchitis virus (IBV) are mainly impacted by mutations in the spike 1 (S1) gene. This study focused on whole genome sequencing of an IBV isolate (IBV/Ck/Can/2558004), which represents strains highly prevalent in Canadian commercial poultry, especially concerning features related to its S1 gene and protein sequences. Based on the phylogeny of the S1 gene, IBV/Ck/Can/2558004 belongs to the GI-17 lineage. According to S1 gene and protein pairwise alignment, IBV/Ck/Can/2558004 had 99.44-99.63% and 98.88-99.25% nucleotide (nt) and deduced amino acid (aa) identities, respectively, with five Canadian Delmarva (DMV/1639) IBVs isolated in 2019, and it also shared 96.63-97.69% and 94.78-97.20% nt and aa similarities with US DMV/1639 IBVs isolated in 2011 and 2019, respectively. Further homology analysis of aa sequences showed the existence of some aa substitutions in the hypervariable regions (HVRs) of the S1 protein of IBV/Ck/Can/2558004 compared to US DMV/1639 isolates; most of these variant aa residues have been subjected to positive selection pressure. Predictive analysis of potential N-glycosylation and phosphorylation motifs showed either loss or acquisition in the S1 glycoprotein of IBV/Ck/Can/2558004 compared to S1 of US DMV/1639 IBV. Furthermore, bioinformatic analysis showed some of the aa changes within the S1 protein of IBV/Ck/Can/2558004 have been predicted to impact the function and structure of the S1 protein, potentially leading to a lower binding affinity of the S1 protein to its relevant ligand (sialic acid). In conclusion, these findings revealed that the DMV/1639 IBV isolates are under continuous evolution among Canadian poultry.

Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle.

Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. The balance of the opposing activities of the surface spike (S) and hemagglutinin-esterase (HE) glycoproteins controls BCoV avidity, which is critical for interspecies transmission and host adaptation. Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S. Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12 nucleotide insertion in the receptor-binding domain (RBD) of the HE gene similar to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12 nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and could bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding. The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.

Molecular Analyses of Clinical Isolates and Recombinant SARS-CoV-2 Carrying B.1 and B.1.617.2 Spike Mutations Suggest a Potential Role of Non-Spike Mutations in Infection Kinetics.

Some of the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are less susceptible to neutralization with post-vaccine sera and monoclonal antibodies targeting the viral spike glycoprotein. This raises concerns of disease control, transmissibility, and severity. Numerous substitutions have been identified to increase viral fitness within the nucleocapsid and nonstructural proteins, in addition to spike mutations. Therefore, we sought to generate infectious viruses carrying only the variant-specific spike mutations in an identical backbone to evaluate the impact of spike and non-spike mutations in the virus life cycle. We used en passant mutagenesis to generate recombinant viruses carrying spike mutations of B.1 and B.1.617.2 variants using SARS-CoV-2- bacterial artificial chromosome (BAC). Neutralization assays using clinical sera yielded comparable results between recombinant viruses and corresponding clinical isolates. Non-spike mutations for both variants neither seemed to effect neutralization efficiencies with monoclonal antibodies nor the response to treatment with inhibitors. However, live-cell imaging and microscopy revealed differences, such as persisting syncytia and pronounced cytopathic effect formation, as well as their progression between BAC-derived viruses and clinical isolates in human lung epithelial cell lines and primary bronchial epithelial cells. Complementary RNA analyses further suggested a potential role of non-spike mutations in infection kinetics.

A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection.

COVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections. IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

Preliminary investigation of gene expression levels of PAPP-A, STC-2, and HIF-1α in SARS-Cov-2 infected patients.

BACKGROUND: Coronavirus-19 is still considered a pandemic that influences the world. Other molecular alterations should be clearer besides the increasing cytokine storm and pro-inflammatory molecules. Hypoxic conditions that induce HIF-1α lead to stimulate gene expression of STC-2 that targets PAPP-A expression. This study aimed to determine gene expression levels of PAPP-A, STC-2, and HIF-1α in COVID-19 infection. We also aimed to reveal the relationship of these genes with laboratory and clinical data of COVID-19 patients. MATERIALS AND RESULTS: We extracted RNA from peripheral blood samples of COVID-19(+) and COVID-19(-) individuals. The real-time PCR method was used to measure mRNA expression of PAPP-A, STC-2, and HIF-1α. Gene expression analysis was evaluated by the 2-ΔΔCt method. PAPP-A, STC-2, and HIF-1α mRNA expressions of severe patients were higher than healthy individuals (p = 0.0451, p = 0.4466, p < 0.0001, respectively). Correlation analysis of gene expression patterns of severe patients demonstrated a positive correlation between PAPP-A and STC-2 (p < 0.0001, r = 0.8638). CONCLUSION: This is the first study that investigates the relation of PAPP-A, STC-2, and HIF-1α gene expression in patients with COVID-19 infection. Besides the routine laboratory findings, PAPP-A, STC-2, and HIF-1α mRNA expressions may be considered to patients' prognosis as a sign of increased cytokines and pro-inflammatory molecules.

Genetic Analysis of the Complete S1 Gene in Japanese Infectious Bronchitis Virus Strains.

The complete nucleotide sequence of the S1 glycoprotein gene of the Japanese infectious bronchitis virus (IBV) strains was determined and genetically analyzed. A total of 61 Japanese IBV strains were classified into seven genotypes, namely GI-1, 3, 7, 13, 18, 19, and GVI-1 using the classification scheme that was proposed by Valastro et al, with three exceptions. These genotypes practically corresponded to those defined in Japan, namely Mass, Gray, JP-II, 4/91, JP-I, JP-III, and JP-IV, which have been identified through their partial nucleotide sequences containing hypervariable regions 1 and 2. In addition, three exceptive strains were considered to be derived from recombination within the S1 gene of IBV strains G1-13 and GI-19. By analyzing the amino acid polymorphism of the S1 glycoprotein among Japanese genotypes, a diversity was observed based on the genotype-specific amino acid residue, the proteolytic cleavage motif at the S1/S2 cleavage site, and the position of the potential N-glycosylation sites.

Loss of imprinting of the Igf2-H19 ICR1 enhances placental endocrine capacity via sex-specific alterations in signalling pathways in the mouse.

Imprinting control region (ICR1) controls the expression of the Igf2 and H19 genes in a parent-of-origin specific manner. Appropriate expression of the Igf2-H19 locus is fundamental for normal fetal development, yet the importance of ICR1 in the placental production of hormones that promote maternal nutrient allocation to the fetus is unknown. To address this, we used a novel mouse model to selectively delete ICR1 in the endocrine junctional zone (Jz) of the mouse placenta (Jz-ΔICR1). The Jz-ΔICR1 mice exhibit increased Igf2 and decreased H19 expression specifically in the Jz. This was accompanied by an expansion of Jz endocrine cell types due to enhanced rates of proliferation and increased expression of pregnancy-specific glycoprotein 23 in the placenta of both fetal sexes. However, changes in the endocrine phenotype of the placenta were related to sexually-dimorphic alterations to the abundance of Igf2 receptors and downstream signalling pathways (Pi3k-Akt and Mapk). There was no effect of Jz-ΔICR1 on the expression of targets of the H19-embedded miR-675 or on fetal weight. Our results demonstrate that ICR1 controls placental endocrine capacity via sex-dependent changes in signalling.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Omicron variant (B.1.1.529) of SARS-CoV-2: understanding mutations in the genome, S-glycoprotein, and antibody-binding regions.

The Omicron variant has been detected in nearly 150 countries. We analyzed the mutational landscape of Omicron throughout the genome, focusing the S-glycoprotein. We also evaluated mutations in the antibody-binding regions and observed some important mutations overlapping those of previous variants including N501Y, D614G, H655Y, N679K, and P681H. Various new receptor-binding domain mutations were detected, including Q493K, G496S, Q498R, S477N, G466S, N440K, and Y505H. New mutations were found in the NTD (Δ143-145, A67V, T95I, L212I, and Δ211) including one new mutation in fusion peptide (D796Y). There are several mutations in the antibody-binding region including K417N, E484A, Q493K, Q498R, N501Y, and Y505H and several near the antibody-binding region (S477N, T478K, G496S, G446S, and N440K). The impact of mutations in regions important for the affinity between spike proteins and neutralizing antibodies was evaluated. Furthermore, we examined the effect of significant antibody-binding mutations (K417N, T478K, E484A, and N501Y) on antibody affinity, stability to ACE2 interaction, and possibility of amino acid substitution. All the four mutations destabilize the antibody-binding affinity. This study reveals future directions for developing neutralizing antibodies against the Omicron variant.

Lassa virus glycoprotein complex review: insights into its unique fusion machinery.

Lassa virus (LASV), an arenavirus endemic to West Africa, causes Lassa fever-a lethal hemorrhagic fever. Entry of LASV into the host cell is mediated by the glycoprotein complex (GPC), which is the only protein located on the viral surface and comprises three subunits: glycoprotein 1 (GP1), glycoprotein 2 (GP2), and a stable signal peptide (SSP). The LASV GPC is a class one viral fusion protein, akin to those found in viruses such as human immunodeficiency virus (HIV), influenza, Ebola virus (EBOV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These viruses are enveloped and utilize membrane fusion to deliver their genetic material to the host cell. Like other class one fusion proteins, LASV-mediated membrane fusion occurs through an orchestrated sequence of conformational changes in its GPC. The receptor-binding subunit, GP1, first engages with a host cell receptor then undergoes a unique receptor switch upon delivery to the late endosome. The acidic pH and change in receptor result in the dissociation of GP1, exposing the fusion subunit, GP2, such that fusion can occur. These events ultimately lead to the formation of a fusion pore so that the LASV genetic material is released into the host cell. Interestingly, the mature GPC retains its SSP as a third subunit-a feature that is unique to arenaviruses. Additionally, the fusion domain contains two separate fusion peptides, instead of a standard singular fusion peptide. Here, we give a comprehensive review of the LASV GPC components and their unusual features.

Low-Valent Calix[4]arene Glycoconjugates Based on Hydroxamic Acid Bearing Linkers as Potent Inhibitors in a Model of Ebola Virus Cis-Infection and HCMV-gB-Recombinant Glycoprotein Interaction with MDDC Cells by Blocking DC-SIGN.

In addition to a variety of viral-glycoprotein receptors (e.g., heparan sulfate, Niemann-Pick C1, etc.), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), from the C-type lectin receptor family, plays one of the most important pathogenic functions for a wide range of viruses (e.g., Ebola, human cytomegalovirus (HCMV), HIV-1, severe acute respiratory syndrome coronavirus 2, etc.) that invade host cells before replication; thus, its inhibition represents a relevant extracellular antiviral therapy. We report two novel p-tBu-calixarene glycoclusters 1 and 2, bearing tetrahydroxamic acid groups, which exhibit micromolar inhibition of soluble DC-SIGN binding and provide nanomolar IC50 inhibition of both DC-SIGN-dependent Jurkat cis-cell infection by viral particle pseudotyped with Ebola virus glycoprotein and the HCMV-gB-recombinant glycoprotein interaction with monocyte-derived dendritic cells expressing DC-SIGN. A unique cooperative involvement of sugar, linker, and calixarene core is likely behind the strong avidity of DC-SIGN for these low-valent systems. We claim herein new promising candidates for the rational development of a large spectrum of antiviral therapeutics.

Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins.

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.