scholarly journals Improved SARS-CoV-2 Spike Glycoproteins for Pseudotyping Lentiviral Vectors

2021 ◽  
Vol 1 ◽  
Author(s):  
Paul G. Ayoub ◽  
Arunima Purkayastha ◽  
Jason Quintos ◽  
Curtis Tam ◽  
Lindsay Lathrop ◽  
...  
Keyword(s):  
Viral Entry ◽  
Neutralizing Antibodies ◽  
High Specificity ◽  
Lentiviral Vectors ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
Mutant Form ◽  
Tail Domain ◽  
Surface Receptor

The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the native CD4 tropic envelope protein of the virus and thereby either limit or expand the target cell population. We generated a modified S glycoprotein of SARS-Cov-2 to pseudotype lentiviral vectors which efficiently transduced ACE2-expressing cells with high specificity and contain minimal off-target transduction of ACE2 negative cells. By utilizing optimized codons, modifying the S cytoplasmic tail domain, and including a mutant form of the spike protein, we generated an expression plasmid encoding an optimized protein that produces S-pseudotyped lentiviral vectors at an infectious titer (TU/mL) 1000-fold higher than the unmodified S protein and 4 to 10-fold more specific than the widely used delta-19 S-pseudotyped lentiviral vectors. S-pseudotyped replication-defective lentiviral vectors eliminate the need for biosafety-level-3 laboratories required when developing therapeutics against SARS-CoV-2 with live infectious virus. Furthermore, S-pseudotyped vectors with high activity and specificity may be used as tools to understand the development of immunity against SARS-CoV-2, to develop assays of neutralizing antibodies and other agents that block viral binding, and to allow in vivo imaging studies of ACE2-expressing cells.

In Vivo Gene Delivery by Lentiviral Vectors

1999 ◽  
Vol 82 (08) ◽  
pp. 552-554 ◽  
Author(s):  
Luigi Naldini
Keyword(s):  
Viral Entry ◽  
State Level ◽  
Lentiviral Vectors ◽  
Retroviral Vectors ◽  
Target Cells ◽  
Therapeutic Gene ◽  
Retroviral Infection ◽  
Nuclear Localization Signals ◽  
Nucleoprotein Complexes

IntroductionSuccessful gene therapy requires the efficient delivery and sustained expression of a therapeutic gene into the tissues of a human body. Most of the candidate tissues for therapeutic gene transfer are made of quiescent cells, such as from the brain, liver, and muscle. Thus, the optimal vector should infect nondividing cells, become stably associated with the genome of target cells, and support a high, steady-state level of transcription.1,2 Like all vectors derived from retroviruses, lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes. Both of these features are important for achieving sustained expression of the transgene. Moreover, lentiviral vectors infect nondividing cells, a feature sharply distinguishing them from simple or onco-retroviral vectors.3 Upon infection, retroviruses deliver a nucleoprotein complex that reverse transcribes the viral RNA and integrates the newly made DNA into the chromatin. The nucleoprotein complexes of onco-retroviruses are excluded by the nucleus, and they reach the chromatin only when the nuclear membrane is fragmented during mitosis. This explains the dependence of a productive onco-retroviral infection on cell division occurring shortly after viral entry.4,5 In contrast, the nucleoprotein complexes of lentiviruses contain nuclear localization signals that mediate their active transport through the nucleopores during interphase. This explains the capacity of lentiviruses to infect macrophages, a nondividing cell type.6-9

scholarly journals Endocytic Delivery of Vancomycin Mediated by a Synthetic Cell Surface Receptor:  Rescue of Bacterially Infected Mammalian Cells and Tissue Targeting In Vivo

2007 ◽  
Vol 129 (2) ◽  
pp. 268-269 ◽  
Author(s):  
Siwarutt Boonyarattanakalin ◽  
Jianfang Hu ◽  
Sheryl A. Dykstra-Rummel ◽  
Avery August ◽  
Blake R. Peterson
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Cell Surface Receptor ◽  
Synthetic Cell ◽  
Tissue Targeting ◽  
Surface Receptor

scholarly journals Porcine Breast Extracellular Matrix Hydrogel for Spatial Tissue Culture

2018 ◽  
Vol 19 (10) ◽  
pp. 2912 ◽  
Author(s):  
Girdhari Rijal ◽  
Jing Wang ◽  
Ilhan Yu ◽  
David Gang ◽  
Roland Chen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Human Mammary Epithelial Cell ◽  
Tumor Formation ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Breast Diseases ◽  
Porous Scaffolds ◽  
Ecm Proteins ◽  
Surface Receptor

Porcine mammary fatty tissues represent an abundant source of natural biomaterial for generation of breast-specific extracellular matrix (ECM). Here we report the extraction of total ECM proteins from pig breast fatty tissues, the fabrication of hydrogel and porous scaffolds from the extracted ECM proteins, the structural properties of the scaffolds (tissue matrix scaffold, TMS), and the applications of the hydrogel in human mammary epithelial cell spatial cultures for cell surface receptor expression, metabolomics characterization, acini formation, proliferation, migration between different scaffolding compartments, and in vivo tumor formation. This model system provides an additional option for studying human breast diseases such as breast cancer.

scholarly journals Extracellular interactions and ligand degradation shape the nodal morphogen gradient

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Yin Wang ◽  
Xi Wang ◽  
Thorsten Wohland ◽  
Karuna Sampath
Keyword(s):  
Correlation Spectroscopy ◽  
Cell Surface Receptor ◽  
Morphogen Gradient ◽  
Axis Formation ◽  
Cellular Interactions ◽  
Fluorescence Correlation ◽  
Selective Ligand ◽  
Surface Receptor ◽  
First Time

The correct distribution and activity of secreted signaling proteins called morphogens is required for many developmental processes. Nodal morphogens play critical roles in embryonic axis formation in many organisms. Models proposed to generate the Nodal gradient include diffusivity, ligand processing, and a temporal activation window. But how the Nodal morphogen gradient forms in vivo remains unclear. Here, we have measured in vivo for the first time, the binding affinity of Nodal ligands to their major cell surface receptor, Acvr2b, and to the Nodal inhibitor, Lefty, by fluorescence cross-correlation spectroscopy. We examined the diffusion coefficient of Nodal ligands and Lefty inhibitors in live zebrafish embryos by fluorescence correlation spectroscopy. We also investigated the contribution of ligand degradation to the Nodal gradient. We show that ligand clearance via degradation shapes the Nodal gradient and correlates with its signaling range. By computational simulations of gradient formation, we demonstrate that diffusivity, extra-cellular interactions, and selective ligand destruction collectively shape the Nodal morphogen gradient.

scholarly journals Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

2020 ◽  
Author(s):  
Alexandra C. Walls ◽  
Young-Jun Park ◽  
M. Alexandra Tortorici ◽  
Abigail Wall ◽  
Andrew T. McGuire ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Furin Cleavage ◽  
Humoral Immune ◽  
Binding Domains ◽  
Furin Cleavage Site ◽  
Recent Emergence ◽  
Novel Coronavirus

SUMMARYThe recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

scholarly journals The role of Neuropilin-1 in COVID-19

2021 ◽  
Vol 17 (1) ◽  
pp. e1009153
Author(s):  
Bindu S. Mayi ◽  
Jillian A. Leibowitz ◽  
Arden T. Woods ◽  
Katherine A. Ammon ◽  
Alphonse E. Liu ◽  
...  
Keyword(s):  
Immune Function ◽  
Viral Entry ◽  
Axonal Guidance ◽  
Cell Surface Receptor ◽  
Neuropilin 1 ◽  
Surface Receptor ◽  
The Central Nervous System

Neuropilin-1 (NRP-1), a member of a family of signaling proteins, was shown to serve as an entry factor and potentiate SARS Coronavirus 2 (SARS-CoV-2) infectivity in vitro. This cell surface receptor with its disseminated expression is important in angiogenesis, tumor progression, viral entry, axonal guidance, and immune function. NRP-1 is implicated in several aspects of a SARS-CoV-2 infection including possible spread through the olfactory bulb and into the central nervous system and increased NRP-1 RNA expression in lungs of severe Coronavirus Disease 2019 (COVID-19). Up-regulation of NRP-1 protein in diabetic kidney cells hint at its importance in a population at risk of severe COVID-19. Involvement of NRP-1 in immune function is compelling, given the role of an exaggerated immune response in disease severity and deaths due to COVID-19. NRP-1 has been suggested to be an immune checkpoint of T cell memory. It is unknown whether involvement and up-regulation of NRP-1 in COVID-19 may translate into disease outcome and long-term consequences, including possible immune dysfunction. It is prudent to further research NRP-1 and its possibility of serving as a therapeutic target in SARS-CoV-2 infections. We anticipate that widespread expression, abundance in the respiratory and olfactory epithelium, and the functionalities of NRP-1 factor into the multiple systemic effects of COVID-19 and challenges we face in management of disease and potential long-term sequelae.

scholarly journals A ligand-based system for receptor-specific delivery of proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mariano Maffei ◽  
Chiara Morelli ◽  
Ellie Graham ◽  
Stefano Patriarca ◽  
Laura Donzelli ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Protein Delivery ◽  
Genetic Diseases ◽  
Cell Surface Receptor ◽  
Viral Gene ◽  
Promising Alternative ◽  
Large Proteins ◽  
Reporter Mice ◽  
Surface Receptor

AbstractGene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand’s cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

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

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1457
Author(s):  
Maria Fernanda Lay Mendoza ◽  
Marissa Danielle Acciani ◽  
Courtney Nina Levit ◽  
Christopher Santa Maria ◽  
Melinda Ann Brindley
Keyword(s):  
Real Time ◽  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Cell Surface Receptor ◽  
Enveloped Virus ◽  
Glycoprotein G ◽  
Viral Glycoproteins ◽  
Surface Receptor ◽  
Vesicular Stomatitis ◽  
Time Required

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

scholarly journals Extracellular vesicles from symbiotic vaginal lactobacilli inhibit HIV-1 infection of human tissues

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Rogers A. Ñahui Palomino ◽  
Christophe Vanpouille ◽  
Luca Laghi ◽  
Carola Parolin ◽  
Kamran Melikov ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Viral Entry ◽  
Ex Vivo ◽  
Target Cells ◽  
Isolated Cells ◽  
Viral Attachment ◽  
Vaginal Lactobacilli ◽  
Male To Female ◽  
Hiv 1

AbstractThe vaginal microbiota, dominated by Lactobacillus spp., plays a key role in preventing HIV-1 transmission. Here, we investigate whether the anti-HIV effect of lactobacilli is mediated by extracellular vesicles (EVs) released by these bacteria. Human cervico-vaginal and tonsillar tissues ex vivo, and cell lines were infected with HIV-1 and treated with EVs released by lactobacilli isolated from vaginas of healthy women. EVs released by L. crispatus BC3 and L. gasseri BC12 protect tissues ex vivo and isolated cells from HIV-1 infection. This protection is associated with a decrease of viral attachment to target cells and viral entry due to diminished exposure of Env that mediates virus-cell interactions. Inhibition of HIV-1 infection is associated with the presence in EVs of several proteins and metabolites. Our findings demonstrate that the protective effect of Lactobacillus against HIV-1 is, in part, mediated by EVs released by these symbiotic bacteria. If confirmed in vivo, this finding may lead to new strategies to prevent male-to-female sexual HIV-1 transmission.

scholarly journals cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

2001 ◽  
Vol 75 (24) ◽  
pp. 12028-12038 ◽  
Author(s):  
Benhur Lee ◽  
George Leslie ◽  
Elizabeth Soilleux ◽  
Una O'Doherty ◽  
Sarah Baik ◽  
...  
Keyword(s):  
Cell Lines ◽  
Viral Entry ◽  
Parental Cell ◽  
Jurkat Cells ◽  
Target Cells ◽  
Parental Cell Line ◽  
Dependent Manner ◽  
Immunodeficiency Virus ◽  
T Cell Lines

ABSTRACT DC-SIGN is a C-type lectin expressed on dendritic cells and restricted macrophage populations in vivo that binds gp120 and acts intrans to enable efficient infection of T cells by human immunodeficiency virus type 1 (HIV-1). We report here that DC-SIGN, when expressed in cis with CD4 and coreceptors, allowed more efficient infection by both HIV and simian immunodeficiency virus (SIV) strains, although the extent varied from 2- to 40-fold, depending on the virus strain. Expression of DC-SIGN on target cells did not alleviate the requirement for CD4 or coreceptor for viral entry. Stable expression of DC-SIGN on multiple lymphoid lines enabled more efficient entry and replication of R5X4 and X4 viruses. Thus, 10- and 100-fold less 89.6 (R5/X4) and NL4–3 (X4), respectively, were required to achieve productive replication in DC-SIGN-transduced Jurkat cells when compared to the parental cell line. In addition, DC-SIGN expression on T-cell lines that express very low levels of CCR5 enabled entry and replication of R5 viruses in a CCR5-dependent manner, a property not exhibited by the parental cell lines. Therefore, DC-SIGN expression can boost virus infection in cis and can expand viral tropism without affecting coreceptor preference. In addition, coexpression of DC-SIGN enabled some viruses to use alternate coreceptors like STRL33 to infect cells, whereas in its absence, infection was not observed. Immunohistochemical and confocal microscopy data indicated that DC-SIGN was coexpressed and colocalized with CD4 and CCR5 on alveolar macrophages, underscoring the physiological significance of these cis enhancement effects.

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