Development of a novel PCV2 and PCV3 vaccine using virus-like vesicles incorporating Venezuelan equine encephalomyelitis virus-containing vesicular stomatitis virus glycoprotein.

Porcine circovirus disease (PCV) causes substantial economic losses in the pig industry, primarily from porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3). Novel vaccines are necessary to prevent and control PCV infections. PCV coat proteins are crucial for eliciting immunogenic proteins that induce the production of antibodies and immune responses. A vaccine platform utilizing Semliki Forest virus RNA replicons expressing vesicular stomatitis virus glycoprotein (VSV-G), was recently developed. This platform generates virus-like vesicles (VLVs) containing VSV-G exclusively, excluding other viral structural proteins. In our study, we developed a novel virus-like vesicle vaccine by constructing recombinant virus-like vesicles (rVLVs) that also express EGFP. These rVLVs were created using the RNA replicon of Venezuelan equine encephalomyelitis (VEEV) and New Jersey serotype VSV-G. The rVLVs underwent characterization and safety evaluation in vitro. Subsequently, rVLVs expressing PCV2d-Cap and PCV3-Cap proteins were constructed. Immunization of C57 mice with these rVLVs led to a significant increase in anti-porcine circovirus type 2 and type 3 capsid protein antibodies in mouse serum. Additionally, a cellular immune response was induced, as evidenced by high production of IFN-γ and IL-4 cytokines. Overall, this study demonstrates the feasibility of developing a novel porcine circovirus disease vaccine based on rVLVs.

Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics.

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

The fluorescence mKate2 labeling as a visualizing system for monitoring small extracellular vesicles.

Small extracellular vesicles (sEVs) are nanosized vesicles enclosed in a lipid membrane released by nearly all cell types. sEVs have been considered as reliable biomarkers for diagnostics and effective carriers. Despite the clear importance of sEV functionality, sEV research faces challenges imposed by the small size and precise imaging of sEVs. Recent advances in live and high-resolution microscopy, combined with efficient labeling strategies, enable us to investigate the composition and behavior of EVs within living organisms. Here, a modified sEVs was generated with a near infrared fluorescence protein mKate2 using a VSVG viral pseudotyping-based approach for monitoring sEVs. An observed was made that the mKate2-tagged protein can be incorporated into the membranes of sEVs without altering their physical properties. In vivo imaging demonstrates that sEVs labeled with mKate2 exhibit excellent brightness and high photostability, allowing the acquisition of long-term investigation comparable to those achieved with mCherry labeling. Importantly, the mKate2-tagged sEVs show a low toxicity and exhibit a favorable safety profile. Furthermore, the co-expression of mKate2 and rabies virus glycoprotein (RVG) peptide on sEVs enables brain-targeted visualization, suggesting the mKate2 tag does not alter the biodistribution of sEVs. Together, the study presents the mKate2 tag as an efficient tracker for sEVs to monitor tissue-targeting and biodistribution in vivo.

Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole-genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole-genome screen and counter-screen strategy based on a pseudoviral platform that allowed identification of genes specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and vesicular stomatitis virus glycoprotein (VSV-G) mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed the identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein packaged into the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed fluorescent protein reporter upon entry  reduced the number of gene hits and increase specificity for viral entry. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and low-density lipoprotein receptors, respectively, and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry-associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression. Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication.

CD90-targeted lentiviral vectors for HSC gene therapy.

Hematopoietic stem cell (HSC) gene therapy is currently performed on CD34+ hematopoietic stem and progenitor cells containing less than 1% true HSCs and requiring a highly specialized infrastructure for cell manufacturing and transplantation. We have previously identified the CD34+CD90+ subset to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, we developed novel targeted viral vectors to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSV-G-pseudotyped lentiviral vectors that were knocked out for native targeting. We further developed a custom hydrodynamic titration methodology to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer, maintained their ability to fuse with the target cells, and showed highly specific recognition of CD90 on cells ex vivo. Most important, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. Our novel HSC-targeted viral vectors have the potential to significantly enhance the feasibility of ex vivo gene therapy and pave the way for future in vivo applications.

Glycolipid transfer protein knockout disrupts vesicle trafficking to the plasma membrane.

The glycolipid transfer protein (GLTP) has been linked to many cellular processes aside from its best-known in vitro function as a lipid transport protein. It has been proposed to act as a sensor and regulator of glycosphingolipid homeostasis in cells. Furthermore, through its previously determined interaction with the endoplasmic reticulum membrane protein VAP-A (vesicle-associated membrane protein-associated protein A), GLTP may also be involved in facilitating vesicular transport in cells. In this study, we characterized the phenotype of CRISPR/Cas9-mediated GLTP KO HeLa cells. We showed that motility, three-dimensional growth, and cellular metabolism were all altered by GLTP knockout. Expression of a GLTP mutant incapable of binding VAP disrupted cell spheroid formation, indicating that the GLTP-VAP interaction is linked to cellular adhesion, cohesion, and three-dimensional growth. Most notably, we found evidence that GLTP, through its interaction with VAP-A, affects vesicular trafficking, marking the first cellular process discovered to be directly impacted by a change in GLTP expression.

A straightforward microfluidic-based approach toward optimizing transduction efficiency of HIV-1-derived lentiviral vectors in BCP-ALL cells.

Background: HIV-1-derived lentiviral vectors (LVs) are capable of transducing human cells by integrating the transgene into the host genome. In order to do that, LVs should have enough time and space to interact with the surface of the target cells. Herein, we used a microfluidic system to facilitate the transduction of BCP-ALL cells. Methods and Results: We used a SU-8 mold to fabricate a PDMS microfluidic chip containing three channels with a 50 µm height and a surface matching 96-well plates. In order to produce LVs, we used HEK293T cells to package the second generation of LVs. First, we evaluated the cell recovery from the microfluidic chip. Cell recovery assessment showcased that 3 h and 6 h of incubation in microfluidic channels containing 100,000 NALM-6 (BCP-ALL) cells with 2µL of culture media yielded 87±7.2% and 80.6 ± 10% of cell recovery, respectively. Afterward, the effects of LV-induced toxicity were evaluated using 10-30% LV concentrations in time frames ranging from 3 h to 24 h. In 96-well plates, it took 12-24 h for the viruses with 20% and 30% concentrations to affect the cell survival significantly. These effects were intensified in the microfluidic system implying that microfluidic is capable of enhancing LV transduction. Based on the evidence of cell recovery and cell survival we chose 6 h of incubation with 20% LV. Conclusion: The results from EGFP expression showcased that a microfluidic system could increase the LV transduction in BCP-ALL cells by almost 9-folds. All in all, the microfluidic system seems to be a great armamentarium in optimizing LV-based transduction.

Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake.

BACKGROUND: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. METHODS: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. RESULTS: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. CONCLUSION: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.

Synergistic surface-enhanced Raman scattering effect to distinguish live SARS-CoV-2 S pseudovirus.

The COVID-19 pandemic negatively affected the economy and health security on a global scale, causing a drastic change on lifestyle, calling a need to mitigate further transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Surface-enhanced Raman spectroscopy (SERS) has shown great potential in the sensitive and rapid detection of various molecules including viruses, through the identification of characteristic peaks of their outer membrane proteins. Accurate detection can be developed through the synergistic integration effect among SERS-active substrate, the appropriate laser wavelength, and the target analyte. In this study, gold nanocavities (Au NC) and Au nanoparticles upon ZrO2 nano-bowls (Au NPs/pZrO2) were tested and used as SERS-active substrates in detecting SARS-CoV-2 pseudovirus containing S protein as a surface capsid glycoprotein (SARS-CoV-2 S pseudovirus) and vesicular stomatitis virus G (VSV-G) pseudo-type lentivirus (VSV-G pseudovirus) to demonstrate their virus detection capability. The optimized Au NCs and Au NPs/pZrO2 substrates were then verified by examining the repetition of measurement, reproducibility, and detection limit. Due to the difference in geometry and composition of the substrates, the characteristic peak-positions of live SARS-CoV-2 S and VSV-G pseudoviruses in the obtained Raman spectra vary, which were also compared with those of inactivated ones. Based on the experimental results, SERS mechanism of each substrate to detect virus is proposed. The formation of hot spots brought by the synergistic integration effect among substrate, analyte, and laser induction may result differences in the obtained SERS spectra.

Production and Use of Gesicles for Nucleic Acid Delivery.

Over-expression of the vesicular stomatitis virus glycoprotein (VSVG) in mammalian cells can induce the formation of VSVG-pseudotyped vesicles (named "gesicles") from membrane budding. Its use as a nucleic acid delivery tool is still poorly documented. Naked-plasmid DNA can be delivered in animal cells with gesicles in presence of hexadimethrine bromide (polybrene). However, little is known about gesicle manufacturing process and conditions to obtain successful nucleic acid delivery. In this study, gesicles production process using polyethylenimine (PEI)-transfected HEK293 cells was developed by defining the VSVG-plasmid concentration, the DNA:PEI mass ratio, and the time of gesicle harvest. Furthermore, parameters described in the literature relevant for nucleic acid delivery such as (i) component concentrations in assembly mixture, (ii) component addition order, (iii) incubation time, and (iv) polybrene concentration were tested by assessing the transfection capacity of the gesicles complexed with a green fluorescent protein (GFP)-coding plasmid. Interestingly, freezing/thawing cycles and storage at + 4 °C, - 20 °C, and - 80 °C did not reduce gesicles' ability to transfer plasmid DNA. Transfection efficiency of 55% and 22% was obtained for HeLa cells and for hard-to-transfect cells such as human myoblasts, respectively. For the first time, gesicles were used for delivery of a large plasmid (18-kb) with 42% of efficiency and for enhanced green fluorescent protein (eGFP) gene silencing with siRNA (up to 60%). In conclusion, gesicles represent attractive bioreagents with great potential to deliver nucleic acids in mammalian cells.

Reporter gene assay for membrane fusion of extracellular vesicles.

Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by tobacco etch virus protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

Targeting Extracellular Vesicles to Dendritic Cells and Macrophages.

Targeting protein therapeutics to specific cells and tissues is a major challenge in modern medicine. Improving the specificity of protein therapeutic delivery will significantly enhance efficiency in drug development. One of the promising tools for protein delivery is extracellular vesicles (EVs) that are enveloped by a complex lipid bilayer. EVs are secreted by almost all cell types and possess significant advantages: biocompatibility, stability, and the ability to penetrate the blood-brain barrier. Overexpression of the vesicular stomatitis virus protein G (VSV-G) was shown to promote EV formation by the producer cell. We have developed an EV-based system for targeted delivery of protein cargoes to antigen-presenting cells (APCs). In this study, we show that attachment of a recombinant llama nanobody α-CD206 to the N-terminus of a truncated VSV-G increases the selectivity of EV cargo delivery mainly to APCs. These results highlight the outstanding technological and biomedical potential of EV-based delivery systems for correcting the immune response in patients with autoimmune, viral, and oncological diseases.

Orchestrating Extracellular Vesicle With Dual Reporters for Imaging and Capturing in Mammalian Cell Culture.

Background: Recent technological advancements have enabled live-cell imaging of intracellular organelles to monitor their biogenesis in mammalian cells. However, applying this method to gain insight into extracellular organelles, such as extracellular vesicles (EVs), presents unique challenges that require special considerations in design and engineering. Results: We have developed a dual-reporter system that combines genetic fusion, fluorescence microcopy and magnetic beads capture of EVs to study the biogenesis of EVs in mammalian cell cultures. First, we genetically produced a series of reporters by fusing a green fluorescent protein (GFP) and an affinity peptide (6xHis), with either the endogenous transmembrane protein, CD63, or EVs targeting vesicular stomatitis viral glycoprotein (VSVG). Transfection of these reporters into human 293T cells resulted in expression and integration of these reporters into pre-exosome compartments, which were subsequently released into the culture medium. Confocal imaging and nano-particle tracking analysis demonstrated that EVs were appropriately labeled and exhibited a single dominant peak in the 80-110 nm size range, indicating that isolated EVs were comprised of micro-vesicles and/or exosome subpopulations. Incubation of isolated EVs with nickel-coated magnetic beads resulted in successful capture of GFP-positive EVs. Finally, addition of EVs into culture medium was able to reveal the cellular uptake of GFP-labeled EVs by recipient cells. Taken together, our dual-reporter system provides a powerful method for both monitoring and capturing of EVs in mammalian cell culture systems. Conclusion: A dual-reporter system provides a robust tool to study the life cycle of EVs in mammalian cells from biogenesis and excretion to cellular uptake.

Reprogramming Extracellular Vesicles for Protein Therapeutics Delivery.

Delivering protein therapeutics specifically into target cells and tissues is a promising avenue in medicine. Advancing this process will significantly enhance the efficiency of the designed drugs. In this regard, natural membrane-based systems are of particular interest. Extracellular vesicles (EVs), being the bilayer lipid particles secreted by almost all types of cells, have several principal advantages: biocompatibility, carrier stability, and blood-brain barrier penetrability, which make them a perspective tool for protein therapeutic delivery. Here, we evaluate the engineered genetically encoded EVs produced by a human cell line, which allow efficient cargo loading. In the devised system, the protein of interest is captured by self-assembling structures, i.e., "enveloped protein nanocages" (EPN). In their turn, EPNs are encapsulated in fusogenic EVs by the overexpression of vesicular stomatitis virus G protein (VSV-G). The proteomic profiles of different engineered EVs were determined for a comprehensive evaluation of their therapeutic potential. EVs loading mediated by bio-safe Fos-Jun heterodimerization demonstrates an increased efficacy of active cargo loading and delivery into target cells. Our results emphasize the outstanding technological and biomedical potential of the engineered EV systems, including their application in adoptive cell transfer and targeted cell reprogramming.

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors.

BACKGROUND AIMS: Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. METHODS: The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. RESULTS: Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40-50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality. CONCLUSIONS: The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.

Fusogenic Viral Protein-Based Near-Infrared Active Nanocarriers for Biomedical Imaging.

An effective drug delivery system (DDS) relies on an efficient cellular uptake and faster intracellular delivery of theranostic agents, bypassing the endosomal mediated degradation of the payload. The use of viral nanoparticles (VNPs) permits such advancement, as the viruses are naturally evolved to infiltrate the host cells to deliver their genetic material. As a proof of concept, we bioengineered the vesicular stomatitis virus glycoprotein (VSV-G)-based near-infrared (NIR) active viral nanoconstructs (NAVNs) encapsulating indocyanine green dye (ICG) for NIR bioimaging. NAVNs are spherical in size and have the intrinsic cellular-fusogenic properties of VSV-G. Further, the NIR imaging displaying higher fluorescence intensity in NAVNs treated cells suggests enhanced cellular uptake and delivery of ICG by NAVNs compared to the free form of ICG. The overall study highlights the effectiveness of VSV-G-based VNPs as an efficient delivery system for NIR fluorescence imaging.

mTOR controls endoplasmic reticulum-Golgi apparatus trafficking of VSVg in specific cell types.

BACKGROUND: Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway. METHODS: The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts. RESULTS: We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced. CONCLUSIONS: Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction.

BACKGROUND AIMS: Viral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct. METHODS: Assay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations. RESULTS: The limit of detection was 10 copies/µL, whereas negative samples showed <1.3 copies/µL. Within and between assay imprecision coefficients of variation across the reportable range (10-10 000 copies/µL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R2 >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations). CONCLUSIONS: DDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.

Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins.

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.