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Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by excessive proliferation of myeloid cells. The COVID-19 pandemic has raised concerns regarding the impact of SARS-CoV-2 on patients with malignancies, particularly those with CML. This study aimed to investigate the effects of SARS-CoV-2 proteins on CML cell viability and the protective role of vitamin K2 (VK2) in conjunction with imatinib. Experiments conducted on K562 CML cells demonstrated that the SARS-CoV-2 envelope protein induces cytotoxicity and activates caspase 3/7, which are key markers of apoptosis. VK2 mitigated these cytotoxic effects and decreased cytokine production while inhibiting colony formation. Furthermore, the combination of VK2 with imatinib significantly reduced cellular proliferation, diminished mitochondrial membrane potential, and markedly suppressed colony formation. These findings suggest that VK2 protects CML cells from SARS-CoV-2-induced cytotoxicity and enhances the therapeutic efficacy of imatinib, presenting a potential strategy to improve CML treatment during the COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

PDZ (PSD-95 [postsynaptic density protein 95]/Dlg [Discs large]/ZO-1 [zonula occludens-1]) domain–containing proteins constitute a large family of scaffolds involved in a wide range of cellular tasks and are mainly studied in polarity functions. Diverse host PDZ proteins can be targeted by viral pathogens that express proteins containing PDZ-binding motifs (PDZbms). Previously, we have identified host PDZ-based interactions with the SARS-CoV-2 E protein (2E) in human monocytes. Here, we deepen the study of these interactions by docking and molecular dynamics analyses to identify the most favorable PDZ-PDZbm interaction of 7 host PDZ proteins with the PDZbm of 2E. In addition, we analyzed changes in the expression of 3 of the PDZ proteins identified as 2E interactors in monocytes (syntenin, ZO-2, and interleukin-16), in human monocyte-derived macrophages and in dendritic cells upon stimulation. Our results suggest that these PDZ proteins may have important functions in professional antigen-presenting cells, and their targeting by the PDZbm of 2E, a central virulence determinant of SARS-CoV-2, supports the hypothesis that such PDZ-dependent interaction in immune cells may constitute a viral evasion mechanism. An inhibitor design based on the PDZbm of 2E in the development of drugs against a variety of diseases is discussed. [ABSTRACT FROM AUTHOR]

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here, it is reported that the development of small molecule degraders of the envelope (E) protein of dengue virus. Two classes of bivalent E‐degraders are developed by linking two previously reported E‐binding small molecules, GNF‐2, and CVM‐2‐12‐2, to a glutarimide‐based recruiter of the CRL4CRBN ligase to effect proteosome‐mediated degradation of the E protein. ZXH‐2‐107 (based on GNF‐2) is an E‐degrader with ABL inhibitory activity while ZXH‐8‐004 (based on CVM‐2‐12‐2) is a selective and potent E‐degrader. These two compounds provide proof of concept that difficult‐to‐drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class of direct‐acting antiviral drugs. [ABSTRACT FROM AUTHOR]

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles. [ABSTRACT FROM AUTHOR]

Syncytins are envelope genes of retroviral origin that play a critical role in the formation of a syncytial structure at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one observed in all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryonic development. The cellular receptor for SynA has been identified as the membrane protein LY6E and is not the receptor for SynB. Here, by combining a cell-cell fusion assay with the screening of a human ORFeome-derived expression library, we identified the transmembrane multipass sodium-dependent phosphate transporter 1 PiT1/SLC20A1 as the receptor for SynB. Transfection of cells with the cloned receptor, but not the closely related PiT2/SLC20A2, leads to their fusion with cells expressing SynB, with no cross-reactive fusion activity with SynA. The interaction between the two partners was further demonstrated by immunoprecipitation. PiT1/PiT2 chimera and truncation experiments identified the PiT1 N-terminus as the major determinant for SynB-mediated fusion. RT-qPCR analysis of PiT1 expression on a panel of mouse adult and fetal tissues revealed a concomitant increase of PiT1 and SynB specifically in the developing placenta. Finally, electron microscopy analysis of the placenta of PiT1 null embryo before they die (E11.5) disclosed default of ST-II formation with lack of syncytialization, as previously observed in cognate SynB null placenta, and consistent with the present identification of PiT1 as the SynB partner. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) gains genetic mutations during continuous transmission and evolution, making the virus more adaptive and virulent. The clade of DENV-1 genotype I has expanded and become the predominant genotype in Asia and the Pacific areas, but the underlying mechanisms are unclear. A combined analysis of nonsynonymous mutations in domain III of the envelope protein and their biological effects on virus pathogenesis and transmission was evaluated. Phylogenetic analyses found three nonsynonymous mutations (V324I, V351L, and V380I) in domain III of the envelope protein, which emerged in 1970s-1990s and stably inherited and expanded in contemporary strains after 2000. We generated reverse-mutated viruses (I324V, L351V, and I380V) based on an infectious clone of an epidemic DENV-1 strain (NIID02-20), and the results suggested that the infectivity of the contemporary epidemic virus (wild type, WT) has increased compared to the reverse mutant viruses in mammalian hosts but not mosquito vectors. The WT virus showed a higher binding affinity to host cells and increased virion stability. In addition, weaker immunogenicity and higher resistance to neutralizing antibodies of the WT virus indicated a trend of immune escape. The data suggested that nonsynonymous mutations of the E protein (V324I, V351L, and V380I) promote infectivity and immune evasion of DENV-1 genotype I, which may facilitate its onward transmission on a global scale. [ABSTRACT FROM AUTHOR]

The envelope (E) protein is present in all coronavirus genera. This protein can form pentameric oligomers with ion channel activity which have been proposed as a possible therapeutic target. However, high resolution structures of E channels are limited to those of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the recent COVID-19 pandemic. In the present work, we used Alphafold-2 (AF2), in ColabFold without templates, to predict the transmembrane domain (TMD) structure of six E-channels representative of genera alpha-, beta- and gamma-coronaviruses in the Coronaviridae family. High-confidence models were produced in all cases when combining multiple sequence alignments (MSAs) obtained from DeepMSA2. Overall, AF2 predicted at least two possible orientations of the α-helices in E-TMD channels: one where a conserved polar residue (Asn-15 in the SARS sequence) is oriented towards the center of the channel, ‘polar-in’, and one where this residue is in an interhelical orientation ‘polar-inter’. For the SARS models, the comparison with the two experimental models ‘closed’ (PDB: 7K3G) and ‘open’ (PDB: 8SUZ) is described, and suggests a ∼60˚ α-helix rotation mechanism involving either the full TMD or only its N-terminal half, to allow the passage of ions. While the results obtained are not identical to the two high resolution models available, they suggest various conformational states with striking similarities to those models. We believe these results can be further optimized by means of MSA subsampling, and guide future high resolution structural studies in these and other viral channels.

Abstract The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor; however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirm that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.

The envelope (env) protein of SARS-CoV-2, a pivotal component of the viral architecture, plays a multifaceted role in viral assembly, replication, pathogenesis, and ion channel activity. These features make it a significant target for understanding virus–host interactions and developing vaccines to combat COVID-19. Recent structural studies provide valuable insights into the conformational dynamics and membrane topology of the SARS-CoV-2 env protein, shedding light on its functional mechanisms. The strong homology and highly conserved structure of the SARS-CoV-2 env protein shape its immunogenicity and functional characteristics. This study examines the ability of the recombinant SARS-CoV-2 env protein to stimulate an immune response. In this study, recombinant envelope proteins were produced using the baculovirus expression system, and their potential efficacy was evaluated in both in vivo and in vitro models. Our results reveal that the env protein of SARS-CoV-2 stimulates humoral and cellular responses and highlight its potential as a promising vaccine candidate for combating the ongoing pandemic. [ABSTRACT FROM AUTHOR]

The limitations of the current vaccination strategy for the Kyasanur Forest Disease virus (KFDV) underscore the critical need for effective antiviral treatments, highlighting the crucial importance of exploring novel therapeutic approaches through in silico drug design. Kyasanur Forest Disease, caused by KFDV, is a tick-borne disease with a mortality of 3–5% and an annual incidence of 400 to 500 cases. In the early stage of infection, the envelope protein plays a crucial role by facilitating host–virus interactions. The objective of this research is to develop effective antivirals targeting the envelope protein to disrupt the virus–host interaction. In line with this, the 3D structure of the envelope protein was modeled and refined through molecular modeling techniques, and subsequently, ligands were designed via de novo design and pharmacophore screening, yielding 12 potential hits followed by ADMET analysis. The top five candidates underwent geometry optimization and molecular docking. Notably, compounds L4 (SA28) and L3 (CNP0247967) are predicted to have significant binding affinities of −8.91 and −7.58 kcal/mol, respectively, toward the envelope protein, based on computational models. Both compounds demonstrated stability during 200 ns molecular dynamics simulations, and the MM-GBSA binding free-energy values were −85.26 ± 4.63 kcal/mol and −66.60 ± 2.92 kcal/mol for the envelope protein L3 and L4 complexes, respectively. Based on the computational prediction, it is suggested that both compounds have potential as drug candidates for controlling host–virus interactions by targeting the envelope protein. Further validation through in-vitro assays would complement the findings of the present in silico investigations. [ABSTRACT FROM AUTHOR]

Abstract Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here, it is reported that the development of small molecule degraders of the envelope (E) protein of dengue virus. Two classes of bivalent E‐degraders are developed by linking two previously reported E‐binding small molecules, GNF‐2, and CVM‐2‐12‐2, to a glutarimide‐based recruiter of the CRL4CRBN ligase to effect proteosome‐mediated degradation of the E protein. ZXH‐2‐107 (based on GNF‐2) is an E‐degrader with ABL inhibitory activity while ZXH‐8‐004 (based on CVM‐2‐12‐2) is a selective and potent E‐degrader. These two compounds provide proof of concept that difficult‐to‐drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class of direct‐acting antiviral drugs.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a re-emerging pathogen with high morbidity associated to congenital infection. Despite the scientific advances since the last outbreak in the Americas, there are no approved specific treatment or vaccines. As the development of an effective prophylactic approach remains unaddressed, DNA vaccines surge as a powerful and attractive candidate due to the efficacy of sequence optimization in achieving strong immune response. In this study, we developed four DNA vaccine constructs encoding the ZIKV prM/M (premembrane/membrane) and E (envelope) proteins in conjunction with molecular adjuvants. The DNA vaccine candidate (called ZK_DSTP), where the entire membrane-anchoring regions were completely removed, was far more immunogenic compared to their counterparts. Furthermore, inclusion of the tPA-SP leader sequence led to high expression and secretion of the target vaccine antigens, therefore contributing to adequate B cell stimulation. The ZK_DSTP vaccine induced high cellular and humoral response in C57BL/6 adult mice, which included high neutralizing antibody titers and the generation of germinal center B cells. Administration of ZK-DSTP incorporating aluminum hydroxide (Alum) adjuvant led to sustained neutralizing response. In consistency with the high and long-term protective response, ZK_DSTP+Alum protected adult mice upon viral challenge. Collectively, the ZK_DSTP+Alum vaccine formulation advances the understanding of the requirements for a successful and protective vaccine against flaviviruses and is worthy of further translational studies. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) vaccination is the most effective way to prevent JE. Plaque reduction neutralization test (PRNT) as the standard method for potency testing for inactivated JE vaccine could not provide the exact potency value. Envelope (E) protein of JE virus induces the body to create neutralizing antibodies. There is a potential for using the determination of E protein to assess the immunogenicity and efficacy of JE vaccine. In this study, an automatic time-resolved fluoroimmunoassay for detection of E protein in JE vaccine was established as a simple and rapid in vitro potency assay to complement PRNT, including the expression and paired screening of monoclonal antibodies, the establishment of assay method and performance verification. A pair of anti-E protein neutralizing antibodies (L022 and L034) were screened to construct the sandwich detection pattern. After pre-treating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a little detection time and eliminated manual error. The results of the validation experiment met the requirements for quality control. The linear range was from 0.78125 U/mL to 25 U/mL, the sensitivity was 0.01 U/mL, the intra-assay coefficient of variation was less than 5 %, and the inter-assay coefficient of variation was less than 10 %. The recovery from the dilution was between 90 % and 110 %. This present TRFIA shown good stability and effectiveness in quality control for samples related to JE vaccine production. The outcomes demonstrated that the present TRFIA could be an alternative in vitro potency assay in quality control for inactivated JE vaccine.

West Nile virus (WNV) is a mosquito-transmitted flavivirus distributed globally for decades and can cause disease in humans and animals. So far, no WNV vaccine has been licensed for human use. Therefore, the development of novel candidate vaccines and the improvement of vaccination strategies is imperative. As the WNV envelope (E) glycoprotein plays an important role in mediating viral binding to cellular receptors and virus-cell membrane fusion, it is a critical target for the host humoral response. Here, we prepared a recombinant truncated envelope protein of WNV (rWNV-80E) and developed a WNV subunit vaccine formulation with a combination of aluminum hydroxide (alum) and a synthetic oligonucleotide CpG as adjuvants. C57BL/6 mice were immunized twice intramuscularly at 28-day intervals with 5 µg purified rWNV-80E adjuvanted with Alum/CpG. WNV E-specific IgG was detected by enzyme-linked immunosorbent assay and neutralizing antibodies (nAbs) was detected using single-round infectious particles of WNV. Furthermore, T cell immunity was detected by enzyme-linked immunospot assay and intracellular cytokine staining assay. Notably, rWNV-80E was highly immunogenic and elicited potent humoral and cell immunity, as evidenced by significant levels of IFN-γ and TNF-α secretion in the T cells of mice. In summary, the Alum/CpG-adjuvanted rWNV-80E subunit vaccine elicited potent and balanced B- and T-cell immunity in mice, and therefore it is a promising candidate vaccine that warrants further investigation for use in human or veterinary applications.

The envelope (env) protein of SARS-CoV-2, a pivotal component of the viral architecture, plays a multifaceted role in viral assembly, replication, pathogenesis, and ion channel activity. These features make it a significant target for understanding virus–host interactions and developing vaccines to combat COVID-19. Recent structural studies provide valuable insights into the conformational dynamics and membrane topology of the SARS-CoV-2 env protein, shedding light on its functional mechanisms. The strong homology and highly conserved structure of the SARS-CoV-2 env protein shape its immunogenicity and functional characteristics. This study examines the ability of the recombinant SARS-CoV-2 env protein to stimulate an immune response. In this study, recombinant envelope proteins were produced using the baculovirus expression system, and their potential efficacy was evaluated in both in vivo and in vitro models. Our results reveal that the env protein of SARS-CoV-2 stimulates humoral and cellular responses and highlight its potential as a promising vaccine candidate for combating the ongoing pandemic.

The limitations of the current vaccination strategy for the Kyasanur Forest Disease virus (KFDV) underscore the critical need for effective antiviral treatments, highlighting the crucial importance of exploring novel therapeutic approaches through in silico drug design. Kyasanur Forest Disease, caused by KFDV, is a tick-borne disease with a mortality of 3–5% and an annual incidence of 400 to 500 cases. In the early stage of infection, the envelope protein plays a crucial role by facilitating host–virus interactions. The objective of this research is to develop effective antivirals targeting the envelope protein to disrupt the virus–host interaction. In line with this, the 3D structure of the envelope protein was modeled and refined through molecular modeling techniques, and subsequently, ligands were designed via de novo design and pharmacophore screening, yielding 12 potential hits followed by ADMET analysis. The top five candidates underwent geometry optimization and molecular docking. Notably, compounds L4 (SA28) and L3 (CNP0247967) are predicted to have significant binding affinities of −8.91 and −7.58 kcal/mol, respectively, toward the envelope protein, based on computational models. Both compounds demonstrated stability during 200 ns molecular dynamics simulations, and the MM-GBSA binding free-energy values were −85.26 ± 4.63 kcal/mol and −66.60 ± 2.92 kcal/mol for the envelope protein L3 and L4 complexes, respectively. Based on the computational prediction, it is suggested that both compounds have potential as drug candidates for controlling host–virus interactions by targeting the envelope protein. Further validation through in-vitro assays would complement the findings of the present in silico investigations.

Zika virus (ZIKV) is a re-emerging pathogen with high morbidity associated to congenital infection. Despite the scientific advances since the last outbreak in the Americas, there are no approved specific treatment or vaccines. As the development of an effective prophylactic approach remains unaddressed, DNA vaccines surge as a powerful and attractive candidate due to the efficacy of sequence optimization in achieving strong immune response. In this study, we developed four DNA vaccine constructs encoding the ZIKV prM/M (pre-membrane/membrane) and E (envelope) proteins in conjunction with molecular adjuvants. The DNA vaccine candidate (called ZK_ΔSTP), where the entire membrane-anchoring regions were completely removed, was far more immunogenic compared to their counterparts. Furthermore, inclusion of the tPA-SP leader sequence led to high expression and secretion of the target vaccine antigens, therefore contributing to adequate B cell stimulation. The ZK_ΔSTP vaccine induced high cellular and humoral response in C57BL/6 adult mice, which included high neutralizing antibody titers and the generation of germinal center B cells. Administration of ZK-ΔSTP incorporating aluminum hydroxide (Alum) adjuvant led to sustained neutralizing response. In consistency with the high and long-term protective response, ZK_ΔSTP+Alum protected adult mice upon viral challenge. Collectively, the ZK_ΔSTP+Alum vaccine formulation advances the understanding of the requirements for a successful and protective vaccine against flaviviruses and is worthy of further translational studies.

Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses to the poultry industry in China since 2010. However, the potential threat of TMUV to mammals has not been well studied. In this study, a TMUV HB strain isolated from diseased ducks showed high virulence in BALB/c mice inoculated intranasally compared with the reference duck TMUV strain. Further studies revealed that the olfactory epithelium is one pathway for the TMUV HB strain to invade the central nervous system of mice. Genetic analysis revealed that the TMUV HB virus contains two unique residues in E and NS3 proteins (326K and 519T) compared with duck TMUV reference strains. K326E substitution weakens the neuroinvasiveness and neurovirulence of TMUV HB in mice. Remarkably, the TMUV HB strain induced significantly higher levels of IL-1β, IL-6, IL-8, and interferon (IFN)-α/β than mutant virus with K326E substitution in the brain tissue of the infected mice, which suggested that TMUV HB caused more severe inflammation in the mouse brains. Moreover, application of IFN-β to infected mouse brain exacerbated the disease, indicating that overstimulated IFN response in the brain is harmful to mice upon TMUV infection. Further studies showed that TMUV HB upregulated RIG-I and IRF7 more significantly than mutant virus containing the K326E mutation in mouse brain, which suggested that HB stimulated the IFN response through the RIG-I-IRF7 pathway. Our findings provide insights into the pathogenesis and potential risk of TMUV to mammals. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has caused a huge damage to global society, not only affecting health aspect but also economics. The development of effective vaccines and therapeutics has been a critical step in the fight against this pandemic, and much research has been committed to studying the causative virus, SARS-CoV-2. Pseudovirus, a valuable research tool that allows investigating SARS-CoV-2 in a safe and controlled environment, has gained significant attention in recent years. It is a promising tool for examining the behaviour of the viral envelope protein and the development of vaccines and therapeutics. Finding an optimized pseudovirus system is crucial for various scientific and medical research applications, especially in the study of viral infections, immune responses and vaccine development. It plays a crucial role in advancing our understanding of viral diseases to combat with the new variants. This minreview focuses on the concept of pseudovirus systems and factors affecting the pseudovirus yield. [ABSTRACT FROM AUTHOR]

Viral pathogenesis typically involves numerous molecular mechanisms. Protein aggregation is a relatively unknown characteristic of viruses, despite the fact that viral proteins have been shown to form terminally misfolded forms. Zika virus (ZIKV) is a neurotropic one with the potential to cause neurodegeneration. Its protein amyloid aggregation may link the neurodegenerative component to the pathogenicity associated with the viral infection. Therefore, we investigated protein aggregation in the ZIKV proteome as a putative pathogenic route and one of the alternate pathways. We discovered that it contains numerous anticipated aggregation‐prone regions in this investigation. To validate our prediction, we used a combination of supporting experimental techniques routinely used for morphological characterization and study of amyloid aggregates. Several ZIKV proteins and peptides, including the full‐length envelope protein, its domain III (EDIII) and fusion peptide, Pr N‐terminal peptide, NS1 β‐roll peptide, membrane‐embedded signal peptide 2K, and cytosolic region of NS4B protein, were shown to be highly aggregating in our study. Because our findings show that viral proteins can form amyloids in vitro, we need to do a thorough functional study of these anticipated APRs to understand better the role of amyloids in the pathophysiology of ZIKV infection. [ABSTRACT FROM AUTHOR]

The structural proteins located on the SARS-CoV-2 envelope, namely the spike, membrane and envelope protein, play important roles during the entire viral infection cycle. For example, the interaction between the membrane protein and the other structural proteins results in the formation of new virions, while the envelope protein then mediates their secretion. The membrane protein and envelope protein are considered potential drug targets, driving future exploration of their structures and interactions. However, research efforts are complicated as both proteins are membrane-bound and have several functions depending on their tertiary and quaternary structure. Here we review data originating from different sources and explain what is known about the structure and function of these proteins to date. [ABSTRACT FROM AUTHOR]

Key pointsWe report a novel method for the transient expression of SARS-CoV-2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes. Intracellular expression of SARS-CoV-2 E protein increases intra-Golgi pH. By targeting the SARS-CoV-2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH. This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID-19.AbstractThe envelope (E) protein of coronaviruses such as SARS-CoV-1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS-CoV-2 in mammalian cells by directed targeting using a carboxyl-terminal fluorescent protein tag, mKate2. The wild-type SARS-CoV-2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum-Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS-CoV-2 E to the plasma membrane, which enabled biophysical analysis using whole-cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two-electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+ , Cs+ and K+ . The E current is nearly time- and voltage-independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time- and voltage-dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS-CoV-2 in vitro and possible efficacy in treating COVID-19.

The Zika Virus (ZIKV) is an emerging arbovirus of great public health concern, particularly in the Americas after its last outbreak in 2015. There are still major challenges regarding disease control, and there is no ZIKV vaccine currently approved for human use. Among many different vaccine platforms currently under study, the recombinant envelope protein from Zika Virus (rEZIKV) constitutes an alternative option for vaccine development and has great potential for monitoring ZIKV infection and antibody response. This study describes a method to obtain a bioactive and functional rEZIKV using an E. coli expression system, with the aid of a 5-L airlift bioreactor and following an automated fast protein liquid chromatography (FPLC) protocol, capable of obtaining high yields of approximately 20 mg of recombinant protein per liter of bacterium cultures. The purified rEZIKV presented preserved antigenicity and immunogenicity. Our results show that the use of an airlift bioreactor for the production of rEZIKV is ideal for establishing protocols and further research on ZIKV vaccines bioprocess, representing a promising system for the production of a ZIKV envelope recombinant protein-based vaccine candidate. [ABSTRACT FROM AUTHOR]

Coronavirus disease-19 disease originated in China by the end of 2019 and has spread all over the world, with many casualties reported since then. Research for the discovery of effective pharmaceuticals is going on around the world. Pharmaceutical scientists are keenly interested in revealing the capacities of a large number of already existing molecules in molecular and drug databases to fight against coronavirus. We identified several triazole and quinazolinone derivatives from molecular databases that share key pharmacophore features with oxadiazole, a known drug-like compound, and conducted computational analysis to evaluate their ability to bind with the envelope protein of Severe Acute Respiratory Syndrome Coronavirus 2. The molecules were screened for their absorption, distribution, metabolism, excretion properties and drug-likeness using the SwissADME webserver. Out of a large number of molecules investigated, six were reported in this work that showed appreciable binding energy values. In vitro, in vivo toxicological studies and clinical trials have to be conducted to get the complete picture of their efficiency and toxicity. [ABSTRACT FROM AUTHOR]

The structure proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as nucleocapsid protein (N protein) and envelop protein (E protein), are considered to be the critical pro-inflammatory factors in coronavirus disease 2019 (COVID-19). Vitamin K3 has been reported to exert an anti-inflammatory effect. In this study, we investigated the protective effects of vitamin K3 on SARS-CoV-2 N protein induced-endothelial activation and SARS-CoV-2 E protein induced-cell death in THP-1 cells. The results showed that vitamin K3 reduced N protein-induced monocyte adhesion, suppressed the expression of adhesion molecules, and decreased the mRNA levels of pro-inflammatory cytokines in HLMECs. We confirmed that the effects of vitamin K3 on endothelial activation may be related to the inhibition of the NF-κB signal pathway. In addition, vitamin K3 reversed E protein-induced pyroptosis, inhibited NLRP3/GSDMD signal pathway and reduced the mRNA expression of pro-inflammatory cytokines in THP-1 cells. Our results also showed the protective effects of vitamin K3 on the SARS-CoV-2 structural protein-induced THP-1 cells pyroptosis and endothelial activation via NF-κB signaling pathway. These findings suggested that vitamin K3 potently suppressed the inflammatory response to prevent endothelial activation and monocyte pyroptosis induced by SARS-CoV-2 proteins. This may provide a new strategy for the treatment of COVID-19. [ABSTRACT FROM AUTHOR]

The envelope (E) protein is a small polypeptide that can form ion channels in coronaviruses. In SARS coronavirus 2 (SARS-CoV-2), the agent that caused the recent COVID-19 pandemic, and its predecessor SARS-CoV-1, E protein is found in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC), where virion budding takes place. Several reports claim that E protein promotes the formation of "cation-selective channels". However, whether this term represents specificity to certain ions (e.g., potassium or calcium) or the partial or total exclusion of anions is debatable. Herein, we discuss this claim based on the available data for SARS-CoV-1 and -2 E and on new experiments performed using the untagged full-length E protein from SARS-CoV-2 in planar lipid membranes of different types, including those that closely mimic the ERGIC membrane composition. We provide evidence that the selectivity of the E-induced channels is very mild and depends strongly on lipid environment. Thus, despite past and recent claims, we found no indication that the E protein forms cation-selective channels that prevent anion transport, and even less that E protein forms bona fide specific calcium channels. In fact, the E channel maintains its multi-ionic non-specific neutral character even in concentrated solutions of Ca2+ ions. Also, in contrast to previous studies, we found no evidence that SARS-CoV-2 E channel activation requires a particular voltage, high calcium concentrations or low pH, in agreement with available data from SARS-CoV-1 E. In addition, sedimentation velocity experiments suggest that the E channel population is mostly pentameric, but very dynamic and probably heterogeneous, consistent with the broad distribution of conductance values typically found in electrophysiological experiments. The latter has been explained by the presence of proteolipidic channel structures. [ABSTRACT FROM AUTHOR]

The world has faced unprecedented disruptions like global quarantine and the COVID-19 pandemic due to SARS-CoV-2. To combat these unsettling situations, several effective vaccines have been developed and are currently being used. However, the emergence of new variants due to the high mutation rate of SARS-CoV-2 challenges the efficacy of existing vaccines and has highlighted the need for novel vaccines that will be effective against various SARS-CoV-2 variants. In this study, we exploited the four structural proteins of SARS-CoV-2 to execute a potential multi-epitope vaccine against SARS-CoV-2 and its variants. The vaccine was designed by utilizing the antigenic, non-toxic, and non-allergenic B-cell and T-cell epitopes, which were selected from conserved regions of viral proteins. To build a vaccine construct, epitopes were connected through different linkers and an adjuvant was also attached at the start of the construct to enhance the immunogenicity and specificity of the epitopes. The vaccine construct was then screened through the aforementioned filters and it scored 0.6019 against the threshold of 0.4 on VexiJen 2.0 which validates its antigenicity. Toll-like receptors (i.e., TLR2, TLR3, TLR4, TLR5, and TLR8) and vaccine construct were docked by Cluspro 2.0, and TLR8 showed strong interaction with construct having a maximum negative binding energy of − 1577.1 kCal/mole. C-IMMSIM's immune simulations over three doses of the vaccine and iMODS' molecular dynamic simulations were executed to assess the reliability of the docked complexes. The stability of the vaccine construct was evaluated through the physicochemical analyses and the findings suggested that the manufactured vaccine is stable under a wide range of circumstances and can trigger immune responses against various SARS-CoV-2 variants (due to conserved epitopes). However, to strengthen the formulation of the vaccine and assess its safety and effectiveness, additional investigations and studies are required to support the computational data of this research at in-vitro and in-vivo levels. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic virus, is one of the most important causes of human viral encephalitis. JEV relies on various attachment or entry co-factors to enter host cells. Among these co-factors, hTIM-1 has been identified as an attachment factor to promote JEV infection through interacting with phosphatidylserine (PS) on the viral envelope. However, the reasons why JEV prefers to use hTIM-1 over other PS binding receptors are unknown. Here, we demonstrated that hTIM-1 can directly interact with JEV E protein. The interaction between hTIM-1 and JEV relies on specific binding sites, respectively, ND114115 in the hTIM-1 IgV domain and K38 of the E protein. Furthermore, during the early stage of infection, hTIM-1 and JEV are co-internalized into cells and transported into early and late endosomes. Additionally, we found that the hTIM-1 soluble ectodomain protein effectively inhibits JEV infection in vitro. Moreover, hTIM-1-specific antibodies have been shown to downregulate JEV infectivity in cells. Taken together, these findings suggested that hTIM-1 protein directly interacts with JEV E protein and mediates JEV infection, in addition to the PS-TIM-1 interaction. [ABSTRACT FROM AUTHOR]

The rational design of dengue virus (DENV) vaccines requires a detailed understanding of the molecular basis for antibody-mediated immunity. The durably protective antibody response to DENV after primary infection is serotype specific. However, there is an incomplete understanding of the antigenic determinants for DENV type-specific (TS) antibodies, especially for DENV serotype 3, which has only one well-studied, strongly neutralizing human monoclonal antibody (mAb). Here, we investigated the human B cell response in children after natural DENV infection in the endemic area of Nicaragua and isolated 15 DENV3 TS mAbs recognizing the envelope (E) glycoprotein. Functional epitope mapping of these mAbs and small animal prophylaxis studies revealed a complex landscape with protective epitopes clustering in at least 6-7 antigenic sites. Potently neutralizing TS mAbs recognized sites principally in E glycoprotein domains I and II, and patterns suggest frequent recognition of quaternary structures on the surface of viral particles.

Syncytin‐2 is a membrane fusion protein involved in placenta development that is derived from the endogenous retrovirus envelope gene acquired in the common ancestral lineage of New World and Old World monkeys (OWMs). It is known that syncytin‐2 is conserved between apes and OWMs, suggesting its functional importance; however, syncytin‐2 of common marmosets (Callithrix jacchus) exhibits lower fusogenic activity than those of humans and OWMs in human cell lines. To obtain insight into the functional diversity of syncytin‐2 genes in primates, we examined the syncytin‐2 gene in New World monkeys (NWMs). We experimentally evaluated the cell fusion ability of syncytin‐2 in humans, C. jacchus, and tufted capuchins (Sapajus apella). We found that the cell fusion ability of S. apella was lower than that of human syncytin‐2. Chimeric syncytin‐2 constructs revealed that the amino acid differences in the surface unit of S. apella syncytin‐2 were responsible for the weak cell fusion activity. In addition, genomic sequence analyses of syncytin‐2 revealed that the open reading frames (ORFs) of syncytin‐2 were highly conserved in seven apes and 22 OWMs; however, the syncytin‐2 ORFs of three of 12 NWM species were truncated. Our results suggest that syncytin‐2 in several NWMs may be of less importance than in OWMs and apes, and other syncytin‐like genes may be required for placental development in various NWM species.

Background & objectives: Dengue virus (DENV) is the causative agent of dengue fever (DF) and dengue hemorrhagic fever (DHF). It has four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) based on their antigenic properties. Mostly, the immunogenic epitopes are present in the envelope (E) protein of the virus. Heparan sulfate (HS) acts as a receptor and interacts with the E protein of the virus thus facilitating the entry of dengue virus into human cells. This study focuses on epitope prediction on the E protein of the DENV serotype. The non-competitive inhibitors of HS were designed using bioinformatics. Methods: In the present study, epitope prediction of the E protein of DENV serotypes was performed using the ABCpred server and IEDB analysis resource. The interactions of HS and viral E proteins (PDB ID: 3WE1 and PDB ID:1TG8) were evaluated through AutoDock. Subsequently, non-competitive inhibitors were designed to bind the E protein of DENV better than HS. All the docking results were validated by re-docking the ligand-receptor complexes and superimposing them onto their co-crystallized complexes using AutoDock and visualizing them in Discovery Studio. Results: The result predicted B-cell and T-cell epitopes on the E protein of DENV serotypes. The designed HS ligand 1 (non-competitive inhibitor) demonstrated potential binding with the DENV E protein, thereby inhibiting HS-E protein binding. The re-docked complexes were superimposed entirely onto the native co-crystallized complexes (low root mean square deviation values), which validates the docking protocols. Interpretation & conclusion: The identified B-cell and T-cell epitopes of the E protein and non-competitive inhibitors of HS (ligand 1) could be used in the designing of potential drug candidates against the dengue virus.

Zika virus (ZIKV), a family member in the Flavivirus genus, has re-emerged as a global public health concern. The envelope (E) proteins of flaviviruses play a dual role in viral assembly and entry. To identify the key residues of E in virus entry, we generated a ZIKV trans-complemented particle (ZIKVTCP) system, in which a subgenomic reporter replicon was packaged by trans-complementation with expression of CprME. We performed mutagenesis studies of the loop regions that protrude from the surface of the virion in the E ectodomains (DI, DII, DIII). Most mutated ZIKVTCPs exhibited deficient egress. Mutations in DII and in the hinge region of DI and DIII affected prM expression. With a bioorthogonal system, photocrosslinking experiments identified crosslinked intracellular E trimers and demonstrated that egress-deficient mutants in DIII impaired E trimerization. Of these mutants, an E-trimerization-dead mutation D389A that nears the E-E interface between two neighbouring spikes in the immature virion completely abolished viral egress. Several mutations abolished ZIKVTCPs’ entry, without severely affecting viral egress. Further virus binding experiments demonstrated a deficiency of the mutated ZIKVTCPs in virus attachment. Strikingly, synthesized peptide containing residues of two mutants (268-273aa in DII) could bind to host cells and significantly compete for viral attachment and interfere with viral infection, suggesting an important role of these resides in virus entry. Our findings uncovered the requirement for DIII mediated-E trimerization in viral egress, and discovered a key residue group in DII that participates in virus entry.

Thermal response of an envelope protein conformation from coronavirus-2 (CoVE) is studied by a coarse-grained Monte Carlo simulation. Three distinct segments, the N-terminal, Trans-membrane, and C-terminal are verified from its specific contact profile. The radius of gyration (Rg) reveals a non-monotonic sub-universal thermal response: Rg decays substantially on heating in native phase under low-temperature regime in contrast to a continuous increase on further raising the temperature prior to its saturation to a random-coil in denature phase. The globularity index which is a measure of effective dimension of the protein, decreases as the protein denatures from a globular to a random-coil conformation.

Context: Dengue is one of the most common infectious diseases found in tropical and subtropical regions, particularly in urban and semi-urban areas. Management dengue until now has not had a specific therapy. The development of dengue therapy using traditional plants as the main source of dengue therapy is needed. Sterculia quadrifida R. Br stem bark is one of the traditional plants in Indonesia, widely used by local people to treat various diseases. Aims: To identify the potency of S. quadrifida stem bark extract against the envelope protein and NS5 RdRp (RNA-dependent RNA polymerase) in dengue infection through in silico approach. Methods: All ligands from S. quadrifida stem bark extract from the previous study and protein preparations were retrieved from the PubChem and RSCB data bank database, respectively. Drug-likeness using Lipinski’s rule of five methods. Pyrx, PyMOL, and Discovery Studio 2.0 were used to analyze and visualize the potency of S. quadrifida stem bark-specific compounds against the envelope and NS5 RdRp. Results: Epicatechin and scopoletin have the lowest affinity bond, some noncovalent interaction, and also similarity in the position of the amino acid interaction to the reference control ribavirin. Conclusions: Epicatechin and scopoletin from S. quadrifida have antiviral potential against dengue by disposing of envelope protein and NS5 RdRp.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing COVID-19 pandemic that has devastated mankind with an unprecedented impact on both health and economic condition globally. The envelope protein of SARS-CoV-2 is a multifunctional viroporin across endoplasmic reticulum-Golgi intermediate compartment. SARS-CoV-2 envelope (E) protein plays a crucial role in the virus life cycle. The objective of the present study was to identify the residue conservation in the SARS-CoV-2 E protein. The study was based on 2,654,250 amino acid sequences for the E protein. On the whole, this study exposed residues that are universally conserved among different strains of SARS-CoV-2. These universally conserved residues might be involved in either structure stabilizing or protein-protein interactions. The conserved residues identified in the present study in conjunction with structural analysis of the E protein could form the basis for designing universal anti-SARS-CoV-2 drugs which are resistant to mutations arising in the future.

Background and aims: Renal damage in severe coronavirus disease 2019 (COVID-19) is highly associated with mortality. Finding relevant therapeutic candidates that can alleviate it is crucial. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin-receptor blockers (ARBs) have been shown to be harmless to COVID-19 patients, but it remains elusive whether ACEIs/ARBs have protective benefits to them. We wished to determine if ACEIs/ARBs had a protective effect on the renal damage associated with COVID-19, and to investigate the mechanism. Methods: We used the envelope (E) protein of severe acute respiratory syndromecoronavirus-2 (SARS-CoV-2) to induce COVID-19-like multiple organ damage and observed renal fibrosis. We induced the epithelial-mesenchymal transformation of HK-2 cells with E protein, and found that olmesartan could alleviate it significantly. The protective effects of olmesartan on E protein-induced renal fibrosis were evaluated by renal-function assessment, pathologic alterations, inflammation, and the TGF-ß1/Smad2/3 signaling pathway. The distribution of high-mobility group box (HMGB)1 was examined after stimulation with E protein and olmesartan administration. Results: E protein stimulated HMGB1 release, which triggered the immune response and promoted activation of TGF-ß1/Smad2/3 signaling: both could lead to renal fibrosis. Olmesartan regulated the distribution of HMGB1 under E protein stimulation. Olmesartan inhibited the release of HMGB1, and reduced the inflammatory response and activation of TGF-ß1/Smad2/3 signaling. Olmesartan increased the cytoplasmic level of HMGB1 to promote the autophagic degradation of TGF-ß1, thereby alleviating fibrosis further. Conclusion: Olmesartan alleviates E protein-induced renal fibrosis by regulating the release of HMGB1 and its mediated autophagic degradation of TGF-ß1. [ABSTRACT FROM AUTHOR]

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials. [ABSTRACT FROM AUTHOR]

Flaviviruses comprise a number of mosquito‐ or tick‐transmitted human pathogens of global public health importance. Advances in structural biology techniques have contributed substantially to our current understanding of the life cycle of these small enveloped RNA viruses and led to deep insights into details of virus assembly, maturation and cell entry. In addition to large‐scale conformational changes and oligomeric rearrangements of envelope proteins during these processes, there is increasing evidence that smaller‐scale protein dynamics (referred to as virus "breathing") can confer extra flexibility to these viruses for the fine‐tuning of their interactions with the immune system and possibly with cellular factors they encounter in their complex ecological cycles in arthropod and vertebrate hosts. In this review, we discuss how work with tick‐borne encephalitis virus has extended our view on flavivirus breathing, leading to the identification of a novel mechanism of antibody‐mediated infection enhancement and demonstrating breathing intermediates of the envelope protein in the process of membrane fusion. These data are discussed in the context of other flaviviruses and the perspective of a potential role of virus breathing to cope with the requirements of adaptation and replication in evolutionarily very different hosts. [ABSTRACT FROM AUTHOR]

Introduction: The Japanese Encephalitis Virus (JEV) causes the acute inflammatory disease of the central nervous system known as Japanese encephalitis. JEV is a RNA virus (+strand) that is tiny, enclosed, and from the family Flavivirus. The envelope protein (E), which facilitates JEV entrance into the host cell, has been chosen as a possible molecular aim for therapeutic progress in this work. Materials and Methods: The 3D structure of E protein was retrieved from RCSB PDB (id: 3P54). The sdf files of four lead molecules namely etoposide, netropsin, nogalamycin, and novobiocin were downloaded from the PubChem and used for molecular docking against JEV E protein. We then assessed the fluorescence emission intensity of JEV alone and JEV bound to novobiocin at 280–500 nM to confirm the inhibition of JEV by Novobiocin. The secondary structure of JEV was ascertained by measuring its CD spectra. The CD spectra of JEV were examined as a function of temperature to evaluate the protein constancy. Results: Our docking and fluorescence emission spectra results showed that JEV E pro has good binding preference for novobiocin among these four test compounds. Moreover, fluorescence emission spectra of JEV E protein with novobiocin also revealed the 5 µM concentration is an effective novobiocin concentration to inhibit the activity of target protein. One top ranked lead molecule namely novobiocin with strong binding affinity (-8.574 Kcal/mol) to JEV E protein was known based on binding energy. Results of the CD Spectra made it quite evident that there is no random coil. Peaks in the negative range (troughs) at 210–230 nm in the CD spectra indicate that JEV has a secondary structure rich in helices. The data unambiguously show that there is no appreciable variation in the JEV curves. Conclusion: Conclusion of the current work offer a thorough understanding of how the JEV E protein is inhibited and provide information that will help novobiocin be developed as a therapeutic drug against JEV E protein in certain viral diseases. [ABSTRACT FROM AUTHOR]

Background: Although a SARS-CoV-2 vaccine is readily available, new cases of COVID-19 are still occurring. New drug discovery is needed to treat COVID-19. Protein E is one of the potential targets. Two synthetic compounds of bicycloproline derivatives have the potential to be developed. Objective: This study aimed to estimate the interaction of bicycloproline compounds to protein E in-silico. Methods: There were two bicycloproline-derived compounds, MI-09 and MI-30, used in docking. Remdesivir was used as a reference ligand. The crystal structure of the E protein was created using homology modeling, while the test compound was drawn using the Marvin Sketch. MOE 2022.02 and BDS 2021 were used for docking and visualization processes. Results: The pentamer of the SARS-CoV-2 E protein obtained a clash score (1.06); poor rotatomer (0.00%); favored rotamers (98.11%); Ramachandran favored (96.43%); Ramachandran outlier (1.78%); Rama Z-score (-1.08); and mol probity (1.04). Research shows promising inhibition potential of the MI-09 and MI-30. The MI-30 has the best binding energy of -10.3326 kcal/mol. Conclusion: The docking results show that MI-30 has potency as an inhibitor of protein E and can be developed in treating COVID-19. Further research is needed to confirm the result by in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

Avian leukosis virus (ALV) is an oncogenic contagious virus infecting different avian species. This study aims to molecularly detect the circulating strains of ALV in various chicken and duck farms at different Egyptian governorates. Freshly dead birds were exposed for postmortem examination (PM) then samples were processed for histopathological examination as well as molecular detection of ALV using qRT-PCR and sequence analysis of the envelope glycoprotein gene (gp85) surface protein antigen with detection of recombination events that might be found between the detected strains. PM revealed the existence of diffuse enlargement of most internal organs, and in some cases, nodular enlargement was observed. Histopathological investigation showed myeloid cells infiltration of eosinophilic granular cytoplasm in the examined tissues. Five molecularly positive samples were sequenced and submitted in the GenBank with accession numbers MZ614719, MZ614720, MZ614721, MZ614722, and MZ614723. The phylogenetic tree construction based on the sequenced gp85 gene revealed that all Egyptian isolates were nearly arranged in a single branch despite the year of collection and were phylogenetically distant from other sequences. In general, studies concerning the genetic diversity of gp85, and the recombination events concluded that the ALV-J virus is a wide host range involving both chicken and ducks. Because ALV causes serious financial problems to threaten poultry production and neither vaccines nor treatment tools are now available for ALV prevention and control, periodical molecular monitoring with whole genome sequence and analysis for the circulating strains with ethical eradication of the positive birds are recommended to overcome such a problem. [ABSTRACT FROM AUTHOR]

Emerging infections of mosquito-borne Zika virus (ZIKV) pose an increasing threat to human health, as documented over the recent years in South Pacific islands and the Americas in recent years. To better understand molecular mechanisms underlying the increase in human cases with severe pathologies, we recently demonstrated the functional roles of structural proteins capsid (C), pre-membrane (prM), and envelop (E) of ZIKV epidemic strains with the initiation of viral infection in human cells. Specifically, we found that the C-prM region contributes to permissiveness of human host cells to ZIKV infection and ZIKV-induced cytopathic effects, whereas the E protein is associated with viral attachment and early infection. In the present study, we further characterize ZIKV E proteins by investigating the roles of residues isoleucine 152 (Ile152), threonine 156 (Thr156), and histidine 158 (His158) (i.e., the E-152/156/158 residues), which surround a unique N-glycosylation site (E-154), in permissiveness of human host cells to epidemic ZIKV infection. For comparison purpose, we generated mutant molecular clones of epidemic BeH819015 (BR15) and historical MR766-NIID (MR766) strains that carry each others E-152/156/158 residues, respectively. We observed that the BR15 mutant containing the E-152/156/158 residues from MR766 was less infectious in A549-Dual™ cells than parental virus. In contrast, the MR766 mutant containing E-152/156/158 residues from BR15 displayed increased infectivity. The observed differences in infectivity were, however, not correlated with changes in viral binding onto host-cells or cellular responses to viral infection. Instead, the E-152/156/158 residues from BR15 were associated with an increased efficiency of viral membrane fusion inside infected cells due to conformational changes of E protein that enhance exposure of the fusion loop. Our data highlight an important contribution of E-152/156/158 residues to the early steps of ZIKV infection in human cells.

Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the maternofetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial), with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. In this study, we performed RNA sequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (Mya), being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans-both Felidae and Canidae-that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental maternofetal interface, and conserved among all carnivorans, consistent with their shared endotheliochorial placenta. Hyenas are a remarkable exception, with a highly invasive hemochorial placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial maternofetal interface. In this study, we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.