Application of SORTS, a Novel Gene-Edited Cell Selection Method for HIV Study and Therapy

We have recently developed surface oligopeptide knock-in for rapid target selection (SORTS), a novel method to isolate mammalian cells with gene modifications using FACS-sorting. It relies on CRISPR/Cas9-mediated targeted knock-in of a very short promoterless expression construct (250 bp) comprising a Flag or HA epitope embedded into the smallest GPI-protein CD52, and a polyA signal from the beta-globin. CD52 efficiently delivers the epitope to the cell surface, where it serves as a marker for selection, while polyA terminates transcription and silences target gene expression. Primarily, SORTS was developed to knock out genes encoding intracellular or secreted proteins, which cannot be used as markers for selection of live cells. Using in-frame modification of SORTS, we demonstrated the possibility of HIV-1 provirus inactivation via sorting of GPI-tag positive cells. In order to make the “cured” cells resistant to a subsequent HIV-1 infection, the epitope tag in the CD52 molecule was substituted by one of the fusion inhibitor peptides from the CHR-domain of gp41. We selected a series of cell-surface-expressed, GPI-anchored, C34-based peptides that confer a strong cellular resistance to HIV-1 infection mediated by NL4-3, JRFL, or ZM153 Env. These findings together with a monoclonal antibody raised against the C34 peptide provide an opportunity to generate and select HIV-resistant lymphocytes for a therapeutic goal. SORTS was also adapted to engineer transgenic HIV-1 effector Т cells and to study cell-to-cell transmission. To facilitate transgenesis, we developed a knock-in strategy to express GPI-tag from the intronic region of the human PPP1R12C gene (AAVS1 locus) and delivered FRT sites of recombination into both alleles. In summary, SORTS is a novel instrument to isolate rare cells with precise genomic modifications with broad applications, including HIV biology. This work was supported by the Russian Science Foundation (grant 18-14-00333) and the Russian Foundation for Basic Research (grants 18-29-07052, 18-04-01016).

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Cell Surface-Expressed GPI-Anchored Peptides from the CHR Domain of gp41 Are Potent Inhibitors of HIV-1 Fusion

Proceedings ◽

10.3390/proceedings2020050070 ◽

2020 ◽

Vol 50 (1) ◽

pp. 70

Author(s):

Aleksandra Maslennikova ◽

Dmitriy Komkov ◽

Anastasia Zotova ◽

Dmitriy Mazurov

Keyword(s):

Cell Surface ◽

Target Selection ◽

Peptide Inhibitors ◽

Alternative Methods ◽

Replication Assay ◽

Developed Surface ◽

Helix Mimetics ◽

Or Gene ◽

Latent Hiv ◽

Hiv 1

Current antiretroviral therapy efficiently suppresses viral replication but cannot eliminate latent HIV reservoirs. Moreover, the associated high costs, side effects, and drug resistance have stimulated a need for the development of alternative methods of HIV-1/AIDS treatment, such as peptide inhibitors or gene editing. Recently, we have developed Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a method for the rapid selection of CRISPR/Cas9 gene-edited cells via knock-in of the Flag and HA epitope tags embedded into the shortest GPI-protein, CD52. By targeting the capsid region of the HIV-1 genome, we demonstrate that SORTS can be applied in provirus eradication. However, the cells with inactivated provirus will be susceptible to HIV re-infection. We hypothesized that knocking in one of the peptides from the CHR-domain of gp41, which are known potent inhibitors of HIV-1 fusion, instead of the epitope tag, will provide “post-curable” HIV-1 resistance. While these peptides were extensively studied as soluble substances, their inhibitory effects on HIV after expression on cell surfaces via GPI-anchor are largely unknown. In this study, we established HEK293T/CD4/R5 and Raji/CD4/R5 HIV-1 permissive cell lines that stably expressed one of the gp41 peptides C34, MT-C34, MT-C34-R, and MT34-15D, or alfa-helix mimetics HP23L, p52, and MT-WQ-IDL. For cell surface delivery, the indicated peptides were embedded into the CD52 molecule, and upstream GFP was used to select transformed cells. Using a single-cycle replication assay with the inLuc reporter vector and different Envs, we demonstrated that C34-based GPI-anchored peptides inhibited both cell-free and cell-to-cell HIV-1 infection by at least two orders of magnitude. With the exception of HP23L, the alfa-helix mimetics were less potent inhibitors. Thus, peptides from gp41 associated with lipid rafts and exerted a strong inhibitory activity which can far exceed that determined for soluble peptides, but this should be tested further.

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HIV-1 gp120 induces CD4 association with several molecules on the T cell surface and modulates T cell interaction with endothelium and homing

Immunology Letters ◽

10.1016/s0165-2478(97)86948-9 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 29

Author(s):

M Bragardo

Keyword(s):

T Cell ◽

Cell Surface ◽

Cell Interaction ◽

Hiv 1

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Cell surface expression of the HIV-1 envelope glycoproteins is directed from intracellular CTLA-4-containing regulated secretory granules

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.122696599 ◽

2002 ◽

Vol 99 (12) ◽

pp. 8031-8036 ◽

Cited By ~ 32

Author(s):

L. R. Miranda ◽

B. C. Schaefer ◽

A. Kupfer ◽

Z. Hu ◽

A. Franzusoff

Keyword(s):

Cell Surface ◽

Secretory Granules ◽

Surface Expression ◽

Cell Surface Expression ◽

Envelope Glycoproteins ◽

Hiv 1

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Identification of a novel cell attachment domain in the HIV-1 Tat protein and its 90-kDa cell surface binding protein.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53530-4 ◽

1993 ◽

Vol 268 (7) ◽

pp. 5279-5284

Author(s):

B.S. Weeks ◽

K. Desai ◽

P.M. Loewenstein ◽

M.E. Klotman ◽

P.E. Klotman ◽

...

Keyword(s):

Cell Surface ◽

Cell Attachment ◽

Binding Protein ◽

Tat Protein ◽

Surface Binding ◽

Cell Surface Binding ◽

Hiv 1

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Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures

Scientific Reports ◽

10.1038/s41598-021-88866-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Vida Ungerer ◽

Abel J. Bronkhorst ◽

Priscilla Van den Ackerveken ◽

Marielle Herzog ◽

Stefan Holdenrieder

Keyword(s):

Cancer Cells ◽

Histone Modifications ◽

Chemical Properties ◽

Basic Research ◽

Live Cells ◽

Cycle Phase ◽

Cell Free Dna ◽

Free Dna ◽

High Resolution Analysis ◽

Active Release

AbstractRecent advances in basic research have unveiled several strategies for improving the sensitivity and specificity of cell-free DNA (cfDNA) based assays, which is a prerequisite for broadening its clinical use. Included among these strategies is leveraging knowledge of both the biogenesis and physico-chemical properties of cfDNA towards the identification of better disease-defining features and optimization of methods. While good progress has been made on this front, much of cfDNA biology remains uncharted. Here, we correlated serial measurements of cfDNA size, concentration and nucleosome histone modifications with various cellular parameters, including cell growth rate, viability, apoptosis, necrosis, and cell cycle phase in three different cell lines. Collectively, the picture emerged that temporal changes in cfDNA levels are rather irregular and not the result of constitutive release from live cells. Instead, changes in cfDNA levels correlated with intermittent cell death events, wherein apoptosis contributed more to cfDNA release in non-cancer cells and necrosis more in cancer cells. Interestingly, the presence of a ~ 3 kbp cfDNA population, which is often deemed to originate from accidental cell lysis or active release, was found to originate from necrosis. High-resolution analysis of this cfDNA population revealed an underlying DNA laddering pattern consisting of several oligo-nucleosomes, identical to those generated by apoptosis. This suggests that necrosis may contribute significantly to the pool of mono-nucleosomal cfDNA fragments that are generally interrogated for cancer mutational profiling. Furthermore, since active steps are often taken to exclude longer oligo-nucleosomes from clinical biospecimens and subsequent assays this raises the question of whether important pathological information is lost.

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Cellular Immune Response Induced by DNA Immunization of Mice with Drug Resistant Integrases of HIV-1 Clade A Offers Partial Protection against Growth and Metastatic Activity of Integrase-Expressing Adenocarcinoma Cells

Microorganisms ◽

10.3390/microorganisms9061219 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1219

Author(s):

Maria Isaguliants ◽

Olga Krotova ◽

Stefan Petkov ◽

Juris Jansons ◽

Ekaterina Bayurova ◽

...

Keyword(s):

Oxidative Stress ◽

T Cell ◽

Cell Response ◽

Mammalian Cells ◽

Metastatic Potential ◽

T Cell Response ◽

Dna Immunization ◽

Drug Resistant ◽

Metastatic Activity ◽

Hiv 1

Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209–239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.

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Bacteria as cell surface markers on normal and malignant mammalian cells

Cellular and Molecular Life Sciences ◽

10.1007/bf02136714 ◽

1971 ◽

Vol 27 (11) ◽

pp. 1321-1322 ◽

Cited By ~ 1

Author(s):

P. S. Meadows

Keyword(s):

Cell Surface ◽

Mammalian Cells ◽

Surface Markers ◽

Cell Surface Markers

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SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates

Bioinformatics ◽

10.1093/bioinformatics/btaa092 ◽

2020 ◽

Vol 36 (11) ◽

pp. 3447-3456 ◽

Cited By ~ 2

Author(s):

Matthew Waas ◽

Shana T Snarrenberg ◽

Jack Littrell ◽

Rachel A Jones Lipinski ◽

Polly A Hansen ◽

...

Keyword(s):

Cell Surface ◽

Specific Surface ◽

Web Application ◽

Rank Order ◽

Surface Proteins ◽

Supplementary Information ◽

Live Cells ◽

Specific Marker ◽

Cell Type ◽

Cell Type Specific

Abstract Motivation Cell-type-specific surface proteins can be exploited as valuable markers for a range of applications including immunophenotyping live cells, targeted drug delivery and in vivo imaging. Despite their utility and relevance, the unique combination of molecules present at the cell surface are not yet described for most cell types. A significant challenge in analyzing ‘omic’ discovery datasets is the selection of candidate markers that are most applicable for downstream applications. Results Here, we developed GenieScore, a prioritization metric that integrates a consensus-based prediction of cell surface localization with user-input data to rank-order candidate cell-type-specific surface markers. In this report, we demonstrate the utility of GenieScore for analyzing human and rodent data from proteomic and transcriptomic experiments in the areas of cancer, stem cell and islet biology. We also demonstrate that permutations of GenieScore, termed IsoGenieScore and OmniGenieScore, can efficiently prioritize co-expressed and intracellular cell-type-specific markers, respectively. Availability and implementation Calculation of GenieScores and lookup of SPC scores is made freely accessible via the SurfaceGenie web application: www.cellsurfer.net/surfacegenie. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

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Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

The Journal of Cell Biology ◽

10.1083/jcb.200702151 ◽

2007 ◽

Vol 179 (5) ◽

pp. 1067-1082 ◽

Cited By ~ 54

Author(s):

Valeria R. Caiolfa ◽

Moreno Zamai ◽

Gabriele Malengo ◽

Annapaola Andolfo ◽

Chris D. Madsen ◽

...

Keyword(s):

Cell Membrane ◽

Cell Surface ◽

Plasminogen Activator ◽

Fluorescent Protein ◽

Surface Protein ◽

Basal Membrane ◽

Membrane Dynamics ◽

Live Cells ◽

Cell Surface Protein ◽

Protein Assemblies

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.

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