Envelope Protein Research Articles

Enhanced native chemical ligation by peptide conjugation in trifluoroacetic acid.

Chemical ligation of peptides is increasingly used to generate proteins not readily accessible by recombinant approaches. However, a robust method to ligate "difficult" peptides remains to be developed. Here, we report an enhanced native chemical ligation strategy mediated by peptide conjugation in trifluoroacetic acid (TFA). The conjugation between a carboxyl-terminal peptide thiosalicylaldehyde thioester and a 1,3-dithiol-containing peptide in TFA proceeds rapidly to form a thioacetal-linked intermediate, which is readily converted into the desired native amide bond product through simple postligation treatment. The effectiveness and practicality of the method was demonstrated by the successful synthesis of several challenging proteins, including the SARS-CoV-2 transmembrane Envelope (E) protein and nanobodies. Because of the ability of TFA to dissolve virtually all peptides and prevent the formation of unreactive peptide structures, the method is expected to open new opportunities for synthesizing all families of proteins, particularly those with aggregable or colloidal peptide segments.

A Single-Dose mRNA Vaccine Employing Porous Silica Nanoparticles Induces Robust Immune Responses Against the Zika Virus.

Recently, lipid nanoparticles (LNPs)-based mRNA delivery has been approved by the FDA for SARS-CoV-2 vaccines. However, there are still considerable points for improvement in LNPs. Especially, local administration of LNPs-formulated mRNA can cause off-target translation of mRNA in distal organs which can induce unintended adverse effects. With the hypothesis that large and rigid nanoparticles can be applied to enhance retention of nanoparticles at the injection site, a polyethyleneimine (PEI)-coated porous silica nanoparticles (PPSNs)-based mRNA delivery platform is designed. PPSNs not only facilitate localized translation of mRNA at the site of injection but also prolonged protein expression. It is further demonstrated that the development of a highly efficacious Zika virus (ZIKV) vaccine using mRNA encoding full-length ZIKV pre-membrane (prM) and envelope (E) protein delivered by PPSNs. The ZIKV prME mRNA-loaded PPSNs vaccine elicits robust immune responses, including high levels of neutralizing antibodies and ZIKV E-specific T cell responses in C57BL/6 mice. Moreover, a single injection of prME-PPSNs vaccine provided complete protection against the ZIKV challenge in mice.

Identification of Immunodominant Epitopes of Dengue Virus 2 Envelope and NS1 Proteins: Evaluating the Diagnostic Potential of a Synthetic Peptide.

Dengue is a major infectious disease with potential for outbreaks and epidemics. A specific and sensitive diagnosis is a prerequisite for clinical management of the disease. We designed our study to identify epitopes on theDengue virus (DENV) envelope (E) and non-structural protein 1 (NS1) with potential for diagnosis. Serology and immunoinformatic approaches were employed. We collected DENV-positive, DENV-negative and Japanese encephalitis virus-positive samples from collaborating hospitals in 2019 and 2022-2023. Seropositive peptides in 15-18 mer peptide arrays of E and NS1 proteins of DENV2 were determined by an indirect enzyme-linked immunosorbent assay. B-cell linear and conformational epitopes were predicted using BepiPred2.0 and ElliPro, respectively. A consensus recombinant peptide was designed, synthesised and evaluated for its diagnostic potential using patient sera. Eight peptides of E protein and six peptides of NS1 protein were identified to be the most frequently recognised by Dengue-positive patients. These peptide sequences were compared with B-cell epitope regions and found to be overlapped with predicted B-cell linear and conformational epitopes. EP11 and NSP15 showed a 100% amino acid sequence overlap with B-cell epitopes. EP1 and NSP15 had 14 whereas EP28, EP31, EP60 16, NSP12 and NSP32 had more than 15 interacting interface residues with a neutralising antibody, suggesting a strength of interaction. Interestingly, potential epitopes identified were localised on the surface of proteins as visualised by PyMOL. Validation with a recombined synthetic peptide yielded 92.3% sensitivity and 91.42% specificity. Immunodominant regions identified by serology and computationally predicted epitopes overlapped, thereby showing the robustness of the methodology and the peptide designedfor diagnosis.

Computational Exploration of Potential Pharmacological Inhibitors Targeting the Envelope Protein of the Kyasanur Forest Disease Virus

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.

Seaweed assisted preparation of Fe2O3 nanoparticles and their antiviral studies against White Spot Syndrome Virus (WSSV): In vitro and in silico approach.

In the present study, preparation of Fe2O3 nanoparticles using seaweed Sargassum swartzii extract and characterized using XRD, FTIR, and FE-SEM with EDAX analysis. The crystalline nature of γ-Fe2O3 nanoparticles (hematite powders) were confirmed using XRD. In FT-IR spectra of the Fe2O3 nanoparticles, the major bands at 594 cm−1 and 461 cm−1 observed correspond to the metal‑oxygen (FeO) vibrational modes. The surface morphology of Fe2O3 nanoparticles has been studied by FE-SEM. In vitro toxicity and anti-WSSV activity of Fe2O3 nanoparticles were carried out using the shrimp-insect hybrid cell line (PmLyo-Sf9). The bioactivity of Fe2O3 nanoparticles against the WSSV infected with the PmLyo-SF9 cell line was analyzed through dose-dependent attachment inhibition and virucidal activity at an optimum concentration of 320 μg/mL and confirmed by the expression of viral envelope protein VP28. Further, the computational approach of VP28 envelope protein and Fe2O3 binding affinity was confirmed using molecular docking studies. Hence, the Fe2O3 nanoparticles can be formulated as an anti-viral agent against the aquaculture pathogens in efficient disease control.

Discovery of hepatitis B virus subviral particle biogenesis inhibitors from a bioactive compound library

High levels of hepatitis B virus (HBV) surface antigen (HBsAg) in the blood of chronic HBV carriers are considered to drive the exhaustion of antigen-specific T and B lymphocytes and thus responsible for the persistence of infection. Accordingly, therapeutic elimination of HBsAg may facilitate the activation of adaptive antiviral immune responses against HBV and achieve a functional cure of chronic hepatitis B. We discovered recently that an amphipathic alpha helix spanning W156 to R169 of HBV small envelope (S) protein plays an essential role in the morphogenesis of subviral particles (SVPs) and metabolism of S protein. We thus hypothesized that pharmacological disruption of SVP morphogenesis may induce intracellular degradation of S protein and reduce HBsAg secretion. To identify inhibitors of SVP biogenesis, we screened 4417 bioactive compounds with a HepG2-derived cell line expressing HBV S protein and efficiently secreting small spherical SVPs. The screen identified 24 compounds that reduced intracellular SVPs and secreted HBsAg in a concentration-dependent manner. However, 18 of those compounds inhibited the secretion of HBsAg and HBeAg in HBV replicon transfected HepG2 cells at similar efficiency, suggesting each of those compounds may disrupt a common cellular function required for the synthesis and/or secretion of these viral proteins. Interestingly, lycorine more efficiently inhibited the secretion of HBsAg in HepG2 cells transfected with HBV replicons, HepG2.2.15 cells and HBV infected - HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). The structure activity relationship and antiviral mechanism of lycorine against HBV have been determined.

Recent advances in the study of zika virus structure, drug targets, and inhibitors.

Zika Virus (ZIKV) is a positive-strand RNA virus that can lead to Guillain-Barré syndrome or encephalitis in some individuals and hence presents a serious public health risk. Since the first outbreak of ZIKV in Brazil in 2015, no effective clinical inhibitors have been developed, making the development of effective ZIKV drugs an urgent issue that needs to be addressed. ZIKV belongs to the Flaviviridae family, and its structure includes three structural proteins, namely, capsular (C), premembrane (prM), and envelope (E) proteins, as well as seven nonstructural proteins, namely, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. To provide a reference for the development of future ZIKV drugs, this paper reviews the structure of the ZIKV based on recent literature reports, analyzes the potential therapeutic targets of various proteins, and proposes feasible drug design strategies. Additionally, this paper reviews and classifies the latest research progress on several protease inhibitors, such as E protein inhibitors, NS2B-NS3 inhibitors, and NS5 inhibitors, so that researchers can quickly understand the current status of development and the interconnections among these inhibitors.

Serological immune biomarker for disease severity in dengue-infectedpediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using anenzyme-linked immunoassay assaysystem which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

SARS-CoV-2 envelope protein-derived extracellular vesicles act as potential media for viral spillover.

Extracellular vesicles (EVs) are shown to be a novel viral transmission model capable of increasing a virus's tropism. According to our earlier research, cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or transfected with envelope protein plasmids generate a novel type of EVs that are micrometer-sized and able to encase virus particles. Here, we showed the capacity of these EVs to invade various animals both in vitro and in vivo independent of the angiotensin-converting enzyme 2 receptor. First, via macropinocytosis, intact EVs produced from Vero E6 (monkey) cells were able to enter cells from a variety of animals, including cats, dogs, bats, hamsters, and minks, and vice versa. Second, when given to zebrafish with cutaneous wounds, the EVs showed favorable stability in aqueous environments and entered the fish. Moreover, infection of wild-type (WT) mice with heterogeneous EVs carrying SARS-CoV-2 particles led to a strong cytokine response and a notable amount of lung damage. Conversely, free viral particles did not infect WT mice. These results highlight the variety of processes behind viral transmission and cross-species evolution by indicating that EVs may be possible vehicles for SARS-CoV-2 spillover and raising risk concerns over EVs' potential for viral gene transfer.

Serological immune biomarker for disease severity in dengue-infectedpediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using anenzyme-linked immunoassay assaysystem which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

Targeting the Coronavirus SARS‑CoV‑2’s Envelope, Nucleocapsid, and Spike/ Spike RBD Protein: Computational Insight from Multiple Bioactive Compounds as Potential Anti-Viral Drug Candidates

Global health, social, and economic systems have been seriously threatened by the coronavirus disease (COVID-19) pandemic. In addition to the increasing number of deaths, thousands of COVID-19 survivors continue to experience life-altering illness. This study aimed to evaluate multiple bioactive compounds from various indigenous medicinal plants against the structural proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including the envelope, nucleocapsid, and spike/spike receptor-binding domain (RBD) proteins, in search of potential antiviral drug candidates. Computational analysis was used to screen for binding affinities and assess chemical interactions between ligands and target proteins. The findings revealed the top three potential compounds to bind to the envelope protein (cafestol, kahweol, and ledene), nucleocapsid protein (cafestol, kahweol, and thearubigin), spike protein (tannic acid, eugeniin, and kahweol), and spike RBD protein (kahweol, cafestol, and tannic acid). Moreover, the study identified four types of plants that contain potential bioactive compounds against SARS-CoV-2 structural proteins, including black tea (Camellia sinensis), clove (Syzygium aromaticum), common bean (Phaseolus vulgaris), and star anise (Illicium verum). Interestingly, kahweol exhibited possible binding activity against all four target proteins. This result suggests that bioactive compounds from the listed medicinal plants could potentially be developed into antiviral drugs against COVID-19.

Targeting the Coronavirus SARS‑CoV‑2’s Envelope, Nucleocapsid, and Spike/ Spike RBD Protein: Computational Insight from Multiple Bioactive Compounds as Potential Anti-Viral Drug Candidates

Global health, social, and economic systems have been seriously threatened by the coronavirus disease (COVID-19) pandemic. In addition to the increasing number of deaths, thousands of COVID-19 survivors continue to experience life-altering illness. This study aimed to evaluate multiple bioactive compounds from various indigenous medicinal plants against the structural proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including the envelope, nucleocapsid, and spike/spike receptor-binding domain (RBD) proteins, in search of potential antiviral drug candidates. Computational analysis was used to screen for binding affinities and assess chemical interactions between ligands and target proteins. The findings revealed the top three potential compounds to bind to the envelope protein (cafestol, kahweol, and ledene), nucleocapsid protein (cafestol, kahweol, and thearubigin), spike protein (tannic acid, eugeniin, and kahweol), and spike RBD protein (kahweol, cafestol, and tannic acid). Moreover, the study identified four types of plants that contain potential bioactive compounds against SARS-CoV-2 structural proteins, including black tea (Camellia sinensis), clove (Syzygium aromaticum), common bean (Phaseolus vulgaris), and star anise (Illicium verum). Interestingly, kahweol exhibited possible binding activity against all four target proteins. This result suggests that bioactive compounds from the listed medicinal plants could potentially be developed into antiviral drugs against COVID-19.

Molecular dissection of HERV-W dependent microglial- and astroglial cell polarization

The endogenous retrovirus type W (HERV-W) is a human-specific entity, which was initially discovered in multiple sclerosis (MS) patient derived cells. We initially found that the HERV-W envelope (ENV) protein negatively affects oligodendrogenesis and controls microglial cell polarization towards a myelinated axon associated and damaging phenotype. Such first functional assessments were conducted ex vivo, given the human-specific origin of HERV-W. Recent experimental evidence gathered on a novel transgenic mouse model, mimicking activation and expression of the HERV-W ENV protein, revealed that all glial cell types are impacted and that cellular fates, differentiation, and functions were changed. In order to identify HERV-W-specific signatures in glial cells, the current study analyzed the transcriptome of ENV protein stimulated microglial- and astroglial cells and compared the transcriptomic signatures to lipopolysaccharide (LPS) stimulated cells, owing to the fact that both ligands can activate toll-like receptor-4 (TLR-4). Additionally, a comparison between published disease associated glial signatures and the transcriptome of HERV-W ENV stimulated glial cells was conducted. We, therefore, provide here for the first time a detailed molecular description of specific HERV-W ENV evoked effects on those glial cell populations that are involved in smoldering neuroinflammatory processes relevant for progression of neurodegenerative diseases.

Characterization of an envelope protein 118L in invertebrate iridescent virus 6 (IIV6).

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482bp of linear dsDNA with 215 non-overlapping and putative protein-encoding ORFs. The IIV6 118L ORF is conserved in all sequenced members of the Iridoviridae and encodes a 515 amino acid protein with three predicted transmembrane domains and several N-glycosylation/N-myristoylation sites. In this study, we characterized the 118L ORF by both deleting it from the viral genome and silencing its expression with dsRNA in infected insect cells. The homologous recombination method was used to replace 118L ORF with the green fluorescent protein (gfp) gene. Virus mutants in which the 118L gene sequence had been replaced with gfp were identified by fluorescence microscopy but could not be propagated separately from the wild-type virus in insect cells. Unsuccessful attempts to isolate the mutant virus with the 118L gene deletion suggested that the protein is essential for virus replication. To support this result, we used dsRNA to target the 118L gene and showed that treatment resulted in a 99% reduction in virus titer. Subsequently, we demonstrated that 118L-specific antibodies produced against the 118L protein expressed in the baculovirus vector system were able to neutralize the virus infection. All these results indicate that 118L is a viral envelope protein that is required for the initiation of virus replication.

Quantitative proteome analysis of LAP1-deficient human fibroblasts: A pilot approach for predicting the signaling pathways deregulated in LAP1-associated diseases

Lamina-associated polypeptide 1 (LAP1), a ubiquitously expressed nuclear envelope protein, appears to be essential for the maintenance of cell homeostasis. Although rare, mutations in the human LAP1-encoding TOR1AIP1 gene cause severe diseases and can culminate in the premature death of affected individuals. Despite there is increasing evidence of the pathogenicity of TOR1AIP1 mutations, the current knowledge on LAP1's physiological roles in humans is limited; hence, investigation is required to elucidate the critical functions of this protein, which can be achieved by uncovering the molecular consequences of LAP1 depletion, a topic that remains largely unexplored. In this work, the proteome of patient-derived LAP1-deficient fibroblasts carrying a pathological TOR1AIP1 mutation (LAP1 E482A) was quantitatively analyzed to identify global changes in protein abundance levels relatively to control fibroblasts. An in silico functional enrichment analysis of the mass spectrometry-identified differentially expressed proteins was also performed, along with additional in vitro functional assays, to unveil the biological processes that are potentially dysfunctional in LAP1 E482A fibroblasts. Collectively, our findings suggest that LAP1 deficiency may induce significant alterations in various cellular activities, including DNA repair, messenger RNA degradation/translation, proteostasis and glutathione metabolism/antioxidant response. This study sheds light on possible new functions of human LAP1 and could set the basis for subsequent in-depth mechanistic investigations. Moreover, by identifying deregulated signaling pathways in LAP1-deficient cells, our work may offer valuable molecular targets for future disease-modifying therapies for TOR1AIP1-associated nuclear envelopathies.

Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.

The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors. The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction. The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software. The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation. We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

Analysis of Virus-Specific B Cell Epitopes Reveals Extensive Antigen Degradation Prior to Recognition.

B cell epitopes must be visible for recognition by cognate B cells and/or antibodies. Here, we studied that premise for known linear B cell epitopes that were collected from the Immune Epitope Database as being recognized by humans during microbial infections. We found that the majority of such known B cell epitopes are virus-specific linear B cell epitopes (87.96%), and most are located in antigens that remain enclosed in host cells and/or virus particles, preventing antibody recognition (18,832 out of 29,225 epitopes). Moreover, we estimated that only a minority (32.72%) of the virus-specific linear B cell epitopes that are found in exposed viral regions (e.g., the ectodomains of envelope proteins) are solvent accessible on intact antigens. Hence, we conclude that ample degradation/processing of viral particles and/or infected cells must occur prior to B cell recognition, thus shaping the B cell epitope repertoire.

AcMNPV P74 is cleaved at R325 and R334 by proteinases of both OB and BBMV to expose a potential fusion peptide for oral infection.

Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.

Antiviral activity of pimecrolimus against dengue virus type 2 infection in vitro and in vivo

Dengue virus (DENV) infection is a public health concern in several countries and is associated with severe diseases, such as dengue hemorrhagic fever and dengue shock syndrome. DENVs are transmitted to humans via the bites of infected Aedes mosquitoes, and no antiviral therapeutics are currently available. In this work, we aimed to identify antiviral drugs against DENV type 2 (DENV2) infections and selected pimecrolimus as a potential antiviral drug candidate. Pimecrolimus significantly inhibited DENV2-mediated cell death and replication in vitro. We also confirmed a decrease in the number of plaques formed as well as in the envelope protein levels of DENV2. The time-of-addition and course experiments revealed that pimecrolimus inhibited DENV2 infection during the early stages of the virus replication cycle. In an experimental mouse model, orally administered pimecrolimus alleviated body weight loss and lethality caused by DENV2 infection, which we used as readouts of the drug’s antiviral potency. Furthermore, pimecrolimus significantly inhibited the DENV2 load and ameliorated focal necrosis in the liver and spleen. Taken together, our in vitro and in vivo findings suggest that pimecrolimus is a promising antiviral drug candidate for the treatment of DENV2 infection.

SARS-CoV-2 Viroporin E Induces Ca2+ Release and Neuron Cell Death in Primary Cultures of Rat Hippocampal Cells Aged In Vitro.

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca2+ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca2+ concentration in hippocampal neurons. Ca2+ responses to the E protein are due to Ca2+ release from intracellular stores at the ER. Moreover, E protein-induced Ca2+ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca2+ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca2+, probably acting like a viroporin, thus producing Ca2+ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.