Establishing a Disease-in-a-Dish Model to Study SARS-CoV-2 Infection During Prenatal Development.

Mother-to-fetus transmission of the SARS-CoV-2 virus via the placenta has been reported but cannot readily be studied in pregnant women. This protocol describes an in vitro method to investigate SARS-CoV-2 infection of human embryonic stem cells (hESCs), which are similar to epiblast cells in young postimplantation embryos. First, SARS-CoV-2 viral pseudoparticles, which contain the spike protein and a fluorescent reporter, are incorporated into a lentivirus backbone that is expanded in HEK 293T cells. Then, an infection assay based on hESCs is used with the viral pseudoparticles. An application of the infection assay in therapeutic drug screening is provided. This protocol allows infection of hESCs by SARS-CoV-2 pseudoparticles to be studied in vitro and can be used in conjunction with other assays to understand and potentially prevent infection. hESCs could also be differentiated to study infection in the three germ layers and their fetal cell derivatives. This disease-in-a-dish model could be readily applied to other hESC lines, and to other viral infections, that affect human prenatal development. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing HEK 293T cells for lentiviral vector transfection Support Protocol 1: Visual inspection of transfected HEK 293T cells Basic Protocol 2: Generating viral pseudoparticles Support Protocol 2: Determining viral titer with HEK 293T-ACE2 cells Basic Protocol 3: Plating hESCs for the infection assay Support Protocol 3: Evaluating transduction efficiency.

Regulatory role of endoplasmic reticulum resident chaperone protein ERp29 in anti-murine ß-coronavirus host cell response.

Gap junctional intercellular communication (GJIC) involving astrocytes is important for proper CNS homeostasis. As determined in our previous studies, trafficking of the predominant astrocyte GJ protein, Connexin43 (Cx43), is disrupted in response to infection with a neurotropic murine ß-coronavirus (MHV-A59). However, how host factors are involved in Cx43 trafficking and the infection response is not clear. Here, we show that Cx43 retention due to MHV-A59 infection was associated with increased ER stress and reduced expression of chaperone protein ERp29. Treatment of MHV-A59-infected astrocytes with the chemical chaperone 4-sodium phenylbutyrate increased ERp29 expression, rescued Cx43 transport to the cell surface, increased GJIC, and reduced ER stress. We obtained similar results using an astrocytoma cell line (delayed brain tumor) upon MHV-A59 infection. Critically, delayed brain tumor cells transfected to express exogenous ERp29 were less susceptible to MHV-A59 infection and showed increased Cx43-mediated GJIC. Treatment with Cx43 mimetic peptides inhibited GJIC and increased viral susceptibility, demonstrating a role for intercellular communication in reducing MHV-A59 infectivity. Taken together, these results support a therapeutically targetable ERp29-dependent mechanism where ß-coronavirus infectivity is modulated by reducing ER stress and rescuing Cx43 trafficking and function.

Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery.

The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency. We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro. After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo.

Vibropolyfection: coupling polymer-mediated gene delivery to mechanical stimulation to enhance transfection of adherent cells.

BACKGROUND: With the success of recent non-viral gene delivery-based COVID-19 vaccines, nanovectors have gained some public acceptance and come to the forefront of advanced therapies. Unfortunately, the relatively low ability of the vectors to overcome cellular barriers adversely affects their effectiveness. Scientists have thus been striving to develop ever more effective gene delivery vectors, but the results are still far from satisfactory. Therefore, developing novel strategies is probably the only way forward to bring about genuine change. Herein, we devise a brand-new gene delivery strategy to boost dramatically the transfection efficiency of two gold standard nucleic acid (NA)/polymer nanoparticles (polyplexes) in vitro. RESULTS: We conceived a device to generate milli-to-nanoscale vibrational cues as a function of the frequency set, and deliver vertical uniaxial displacements to adherent cells in culture. A short-lived high-frequency vibrational load (t = 5 min, f = 1,000 Hz) caused abrupt and extensive plasmalemma outgrowths but was safe for cells as neither cell proliferation rate nor viability was affected. Cells took about 1 hr to revert to quasi-naïve morphology through plasma membrane remodeling. In turn, this eventually triggered the mechano-activated clathrin-mediated endocytic pathway and made cells more apt to internalize polyplexes, resulting in transfection efficiencies increased from 10-to-100-fold. Noteworthy, these results were obtained transfecting three cell lines and hard-to-transfect primary cells. CONCLUSIONS: In this work, we focus on a new technology to enhance the intracellular delivery of NAs and improve the transfection efficiency of non-viral vectors through priming adherent cells with a short vibrational stimulation. This study paves the way for capitalizing on physical cell stimulation(s) to significantly raise the effectiveness of gene delivery vectors in vitro and ex vivo.

Mechanisms and challenges of nanocarriers as non-viral vectors of therapeutic genes for enhanced pulmonary delivery.

With the rapid development of biopharmaceuticals and the outbreak of COVID-19, the world has ushered in a frenzy to develop gene therapy. Therefore, therapeutic genes have received enormous attention. However, due to the extreme instability and low intracellular gene expression of naked genes, specific vectors are required. Viral vectors are widely used attributed to their high transfection efficiency. However, due to the safety concerns of viral vectors, nanotechnology-based non-viral vectors have attracted extensive investigation. Still, issues of low transfection efficiency and poor tissue targeting of non-viral vectors need to be addressed. Especially, pulmonary gene delivery has obvious advantages for the treatment of inherited lung diseases, lung cancer, and viral pneumonia, which can not only enhance lung targeting and but also reduce enzymatic degradation. For systemic diseases therapy, pulmonary gene delivery can enhance vaccine efficacy via inducing not only cellular, humoral immunity but also mucosal immunity. This review provides a comprehensive overview of nanocarriers as non-viral vectors of therapeutic genes for enhanced pulmonary delivery. First of all, the characteristics and therapeutic mechanism of DNA, mRNA, and siRNA are provided. Thereafter, the advantages and challenges of pulmonary gene delivery in exerting local and systemic effects are discussed. Then, the inhalation dosage forms for nanoparticle-based drug delivery systems are introduced. Moreover, a series of materials used as nanocarriers for pulmonary gene delivery are presented, and the endosomal escape mechanisms of nanocarriers based on different materials are explored. The application of various non-viral vectors for pulmonary gene delivery are summarized in detail, with the perspectives of nano-vectors for pulmonary gene delivery.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles.

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

mRNA-carrying lipid nanoparticles that induce lysosomal rupture activate NLRP3 inflammasome and reduce mRNA transfection efficiency.

In the last several years, countless developments have been made to engineer more efficient and potent mRNA lipid nanoparticle vaccines, culminating in the rapid development of effective mRNA vaccines against COVID-19. However, despite these advancements and materials approaches, there is still a lack of understanding of the resultant immunogenicity of mRNA lipid nanoparticles. Therefore, a more mechanistic, design-driven approach needs to be taken to determine which biophysical characteristics, especially related to changes in lipid compositions, drive nanoparticle immunogenicity. Here, we synthesized a panel of six mRNA lipid nanoparticle formulations, varying the concentrations of different lipid components and systematically studied their effect on NLRP3 inflammasome activation; a key intracellular protein complex that controls various inflammatory responses. Initial experiments aimed to determine differences in nanoparticle activation of NLRP3 inflammasomes by IL-1ß ELISA, which unveiled that nanoparticles with high concentrations of ionizable lipid DLin-MC3-DMA in tandem with high cationic lipid DPTAP and low cholesterol concentration induced the greatest activation of the NLRP3 inflammasome. These results were further corroborated by the measurement of ASC specks indicative of NLRP3 complex assembly, as well as cleaved gasdermin-D and caspase-1 expression indicating complex activation. We also uncovered these activation profiles to be mechanistically correlated primarily with lysosomal rupturing caused by the delayed membrane disruption capabilities of ionizable lipids until the lysosomal stage, as well as by mitochondrial reactive oxygen species (ROS) production and calcium influx for some of the particles. Therefore, we report that the specific, combined effects of each lipid type, most notably ionizable, cationic lipids, and cholesterol, is a crucial mRNA lipid nanoparticle characteristic that varies the endo/lysosomal rupture capabilities of the formulation and activate NLRP3 inflammasomes in a lysosomal rupture dependent manner. These results provide a more concrete understanding of mRNA lipid Nanoparticle-Associated Molecular Patterns for the activation of molecular-level immune responses and provide new lipid composition design considerations for future mRNA-delivery approaches.

Transient transfection and purification of SARS-CoV-2 spike protein from mammalian cells.

SARS-CoV-2 spike (S) protein ectodomain purification can be challenging, with engineered and natural variations often resulting in lower yields. Here, we present a detailed transfection and purification protocol for the SARS-CoV-2 S ectodomain. We describe how to trace protein yields during purification using highly sensitive and characteristic changes in S ectodomain intrinsic fluorescence upon thermal denaturation. Additionally, we detail several optimized aspects of the purification including timing and temperature. This protocol facilitates consistent, high-quality preparations of the SARS-CoV-2 S ectodomain. For complete details on the use and execution of this protocol, please refer to Stalls et al. (2022), Gobeil et al. (2022), Edwards et al. (2021), and Henderson et al. (2020).

Random Copolymers of Lysine and Isoleucine for Efficient mRNA Delivery.

Messenger RNA (mRNA) is currently of great interest as a new category of therapeutic agent, which could be used for prevention or treatment of various diseases. For this mRNA requires effective delivery systems that will protect it from degradation, as well as allow cellular uptake and mRNA release. Random poly(lysine-co-isoleucine) polypeptides were synthesized and investigated as possible carriers for mRNA delivery. The polypeptides obtained under lysine:isoleucine monomer ratio equal to 80/20 were shown to give polyplexes with smaller size, positive ζ-potential and more than 90% encapsulation efficacy. The phase inversion method was proposed as best way for encapsulation of mRNA into polyplexes, which are based on obtained amphiphilic copolymers. These copolymers showed efficacy in protection of bound mRNA towards ribonuclease and lower toxicity as compared to lysine homopolymer. The poly(lysine-co-isoleucine) polypeptides showed greater than poly(ethyleneimine) efficacy as vectors for transfection of cells with green fluorescent protein and firefly luciferase encoding mRNAs. This allows us to consider obtained copolymers as promising candidates for mRNA delivery applications.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Histidine-enhanced gene delivery systems: The state of the art.

Gene therapy has emerged as a promising tool for treating different intractable diseases, particularly cancer or even viral diseases such as COVID-19 (coronavirus disease 2019). In this context, various non-viral gene carriers are being explored to transfer DNA or RNA sequences into target cells. Here, we review the applications of the naturally occurring amino acid histidine in the delivery of nucleic acids into cells. The biocompatibility of histidine-enhanced gene delivery systems has encouraged their wider use in gene therapy. Histidine-based gene carriers can involve the modification of peptides, dendrimers, lipids or nanocomposites. Several linear polymers, such as polyethylenimine, poly-l-lysine (synthetic) or dextran and chitosan (natural), have been conjugated with histidine residues to form complexes with nucleic acids for intracellular delivery. The challenges, opportunities and future research trends of histidine-based gene deliveries are investigated.

Protease Activated Receptors: A Pathway to Boosting Mesenchymal Stromal Cell Therapeutic Efficacy in Acute Respiratory Distress Syndrome?

Acute Respiratory Distress Syndrome is the most common cause of respiratory failure among critically ill patients, and its importance has been heightened during the COVID-19 pandemic. Even with the best supportive care, the mortality rate in the most severe cases is 40-50%, and the only pharmacological agent shown to be of possible benefit has been steroids. Mesenchymal stromal cells (MSCs) have been tested in several pre-clinical models of lung injury and been found to have significant therapeutic benefit related to: (a) potent immunomodulation; (b) secretion of epithelial and endothelial growth factors; and (c) augmentation of host defense to infection. Initial translational efforts have shown signs of promise, but the results have not yielded the anticipated outcomes. One potential reason is the relatively low survival of MSCs in inflammatory conditions as shown in several studies. Therefore, strategies to boost the survival of MSCs are needed to enhance their therapeutic effect. Protease-activated receptors (PARs) may represent one such possibility as they are G-protein coupled receptors expressed by MSCs and control several facets of cell behavior. This review summarizes some of the existing literature about PARs and MSCs and presents possible future areas of investigation in order to develop potential, PAR-modified MSCs with enhanced therapeutic efficiency.

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.

Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Bemcentinib and Gilteritinib Inhibit Cell Growth and Impair the Endo-Lysosomal and Autophagy Systems in an AXL-Independent Manner.

AXL, a receptor tyrosine kinase from the TAM (TYRO3 AXL and MER) subfamily, and its ligand growth arrest-specific 6 (GAS6) are implicated in pathogenesis of a wide array of cancers, acquisition of resistance to diverse anticancer therapies and cellular entry of viruses. The continuous development of AXL inhibitors for treatment of patients with cancer and COVID-19 underscores the need to better characterize the cellular effects of AXL targeting.In the present study, we compared the cellular phenotypes of CRISPR-Cas9-induced depletion of AXL and its pharmacological inhibition with bemcentinib, LDC1267 and gilteritinib. Specifically, we evaluated GAS6-AXL signaling, cell viability and invasion, the endo-lysosomal system and autophagy in glioblastoma cells. We showed that depletion of AXL but not of TYRO3 inhibited GAS6-induced phosphorylation of downstream signaling effectors, AKT and ERK1/2, indicating that AXL is a primary receptor for GAS6. AXL was also specifically required for GAS6-dependent increase in cell viability but was dispensable for viability of cells grown without exogenous addition of GAS6. Furthermore, we revealed that LDC1267 is the most potent and specific inhibitor of AXL activation among the tested compounds. Finally, we found that, in contrast to AXL depletion and its inhibition with LDC1267, cell treatment with bemcentinib and gilteritinib impaired the endo-lysosomal and autophagy systems in an AXL-independent manner. IMPLICATIONS: Altogether, our findings are of high clinical importance as we discovered that two clinically advanced AXL inhibitors, bemcentinib and gilteritinib, may display AXL-independent cellular effects and toxicity.

Novel suction-based in vivo cutaneous DNA transfection platform.

This work reports a suction-based cutaneous delivery method for in vivo DNA transfection. Following intradermal Mantoux injection of plasmid DNA in a rat model, a moderate negative pressure is applied to the injection site, a technique similar to Chinese báguàn and Middle Eastern hijama cupping therapies. Strong GFP expression was demonstrated with pEGFP-N1 plasmids where fluorescence was observed as early as 1 hour after dosing. Modeling indicates a strong correlation between focal strain/stress and expression patterns. The absence of visible and/or histological tissue injury contrasts with current in vivo transfection systems such as electroporation. Specific utility was demonstrated with a synthetic SARS-CoV-2 DNA vaccine, which generated host humoral immune response in rats with notable antibody production. This method enables an easy-to-use, cost-effective, and highly scalable platform for both laboratorial transfection needs and clinical applications for nucleic acid­based therapeutics and vaccines.

The evaluation of novel oral vaccines based on self-amplifying RNA lipid nanparticles (saRNA LNPs), saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum to neutralize SARS-CoV-2 variants alpha and delta.

The aim of this study was to present and evaluate novel oral vaccines, based on self-amplifying RNA lipid nanparticles (saRNA LNPs), saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum, to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) variants alpha and delta. After invitro evaluation of the oral vaccines on HEK293T/17 cells, we found that saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum could express S-protein at both mRNA and protein levels. In the next step, BALB/c mice were orally vaccinated with saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum at weeks 1 and 3. Importantly, a high titer of IgG and IgA was observed by all of them, sharply in week 6 (P < 0.05). In all study groups, their ratio of IgG2a/IgG1 was upper 1, indicating Th1-biased responses. Wild-type viral neutralization assay showed that the secreted antibodies in vaccinated mice and recovered COVID-19 patients could neutralize SARS-COV-2 variants alpha and delta. After oral administration of oral vaccines, biodistribution assay was done. It was found that all of them had the same biodistribution pattern. The highest concentration of S-protein was seen in the small intestine, followed by the large intestine and liver.

S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies.

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.

Isolation of Primary Human and Rodent Brain Microvascular Endothelial Cells: Culturing, Characterization, and High-Efficiency Transfection.

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.