Journal: bioRxiv

Article Title: A Nanobody Interaction with SARS-CoV-2 Spike Allows the Versatile Targeting of Lentivirus Vectors

doi: 10.1101/2024.06.06.597774

Figure Lengend Snippet: (A) The panel depicts the cellularly expressed Spike protein in blue, with RBD regions as horseshoe shapes. The viral membrane is depicted as pseudotyped with the Tupaia paramyxovirus (TPMV) fusion (F) protein (yellow), associated with a H-Nano chimera of the binding-defective hemaglutinin (H) protein (green) and a nanobody (Nano; red) that binds to the Spike RBD. In this model, binding of the H-Nano protein to Spike activates the TPMV F protein to fuse the viral and cell membranes. (B) The panel depicts an alternative model, in which binding of the viral H-Nano protein to the cellular Spike protein activates Spike to fuse membranes in a TPMV F-independent fashion. (C) β-galactosidase (βGal) reporter lentiviruses were pseudotyped with the binding-defective TPMV H protein plus the TPMV F protein (H+F), or the H-Nano chimeric protein plus F (H-Nano + F), or the H-Nano protein alone (H-Nano). The viruses were used to infect Spike-expressing cells. Infection levels were measured via βGal activities and were normalized to the H-Nano + F levels. Averages and standard deviations are as shown. Note that the H-Nano+F and H-Nano infection levels both were significantly higher than the H+N control (P<0.001). (D) Spike-positive (Spike+) or Spike-negative (Spike-) cells were infected with βGal lentivirus vectors pseudotyped with the wild type (WT) Vesicular stomatitis virus glycoprotein (VSV G), or H-Nano + F, or H-Nano alone. Infection levels for each pseudotyped virus were normalized to infection of the Spike+ cells, and averages and standard deviations are as shown. Note that the difference for the H-Nano virus between Spike+ and Spike-cells was highly significant (P<0.001), while the difference for H-Nano + F was significant (P=0.002).

Article Snippet: The wild type (WT) Vesicular stomatitis virus glycoprotein (VSV G) expression plasmid (pMD.G), the Tupaia paramyxovirus (TPMV) fusion and hemaglutinin expression plasmids (pCG-TPMV-Fd32, pCG-TPMV-Hd32), the SARS-CoV-2 Spike expression plasmid (CoV2-Spike-D614G), the green fluorescent protein expression plasmid (GFP-NoPS), and the lentivirus GagPol packaging plasmid (psPAX2) all were obtained from Addgene, and were the gifts of Simon Davis, Jakob Reiser, Jennifer Doudna, and Didier Trono ( , – ).

Techniques: Membrane, Binding Assay, Expressing, Infection, Control, Virus

Journal: STAR protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model.

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Figure 2. Monitoring the generation of latently infected Th17 cells upon infection with VSVG-pseudotyped packaged virus of the d2EGFP single- round viral construct Top panel, Dual assessment of the percentage of EGFP-positive cells, which indicates the extent of proviral gene expression, and their cell viability based on propidium iodide (PI) staining. Bottom panel, Corresponding forward/side scatter plots. pi: post-infection; ps: post-shutdown.

Article Snippet: Continued REAGENT or RESOURCE SOURCE IDENTIFIER IL-7 PeproTech 200-07 IL-8 PeproTech 200-08M 1L-10 PeproTech 200-10 IL-23 PeproTech 200-23 TGF-b PeproTech 100-21C Anti-human IL-4 PeproTech 500-M04 Anti-human IFN-g PeproTech 500-M90 Concanavalin A MilliporeSigma C0412 Critical commercial assays Human CD4 naive T cell isolation kit BioLegend 480042 Mouse CD8a positive selection kit STEMCELL 18953 Pacific Blue antibody labeling kit Thermo Fisher Scientific P30013 Alexa Fluor 488 antibody labeling kit Thermo Fisher Scientific A20181 Alexa Fluor 555 antibody labeling kit Thermo Fisher Scientific A20187 Alexa Fluor 647 antibody labeling kit Thermo Fisher Scientific A20186 Experimental models: cell lines HEK293T ATCC CRL-3216 Experimental models: Organisms/strains Polarized and expanded primary Th17 cells This paper N/A Recombinant DNA pHR’-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 pHR’-CD8a-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 Lentiviral gag/pol pCMV-Delta 8.91r Dobrowolski et al.2 Dobrowolski et al.2 pMD.G VSVG Addgene #8454 Other Optima L-100K ultracentrifuge Beckman Coulter N/A SW32 Ti rotor Beckman Coulter N/A BD LSR Fortessa BD Biosciences N/A EasySep magnetic tube holder STEMCELL 18000 Eppendorf 5810R benchtop centrifuge Eppendorf 022625101 STAR Protocols 5, 103398, December 20, 2024 3 Day 2: Generation of single-round VSVG-pseudotyped HIV 2.

Techniques: Infection, Virus, Construct, Gene Expression, Staining

Journal: STAR protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model.

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Figure 3. Tat/Nef dual immunofluorescence flow cytometry of cells infected with VSVG-pseudotyped virus of the d2EGFP single-round viral construct Uninfected, acutely infected, latently infected, and reactivated Th17 cells were immunostained for Tat and Nef as described in the text. The 2D plots show proviral gene expression as monitored by d2EGFP versus Tat (Top), d2EGFP versus Nef (Middle), and Tat versus Nef (Bottom).

Article Snippet: Continued REAGENT or RESOURCE SOURCE IDENTIFIER IL-7 PeproTech 200-07 IL-8 PeproTech 200-08M 1L-10 PeproTech 200-10 IL-23 PeproTech 200-23 TGF-b PeproTech 100-21C Anti-human IL-4 PeproTech 500-M04 Anti-human IFN-g PeproTech 500-M90 Concanavalin A MilliporeSigma C0412 Critical commercial assays Human CD4 naive T cell isolation kit BioLegend 480042 Mouse CD8a positive selection kit STEMCELL 18953 Pacific Blue antibody labeling kit Thermo Fisher Scientific P30013 Alexa Fluor 488 antibody labeling kit Thermo Fisher Scientific A20181 Alexa Fluor 555 antibody labeling kit Thermo Fisher Scientific A20187 Alexa Fluor 647 antibody labeling kit Thermo Fisher Scientific A20186 Experimental models: cell lines HEK293T ATCC CRL-3216 Experimental models: Organisms/strains Polarized and expanded primary Th17 cells This paper N/A Recombinant DNA pHR’-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 pHR’-CD8a-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 Lentiviral gag/pol pCMV-Delta 8.91r Dobrowolski et al.2 Dobrowolski et al.2 pMD.G VSVG Addgene #8454 Other Optima L-100K ultracentrifuge Beckman Coulter N/A SW32 Ti rotor Beckman Coulter N/A BD LSR Fortessa BD Biosciences N/A EasySep magnetic tube holder STEMCELL 18000 Eppendorf 5810R benchtop centrifuge Eppendorf 022625101 STAR Protocols 5, 103398, December 20, 2024 3 Day 2: Generation of single-round VSVG-pseudotyped HIV 2.

Techniques: Cytometry, Infection, Virus, Construct, Gene Expression

Journal: STAR protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model.

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Figure 4. Infection of polarized Th17 cells with VSVG-pseudotyped virus of the CD8a-d2EGFP single-round viral construct and their subsequent positive selection by anti-CD8a sorting In this experiment, cells were CD8a-selected at Day 5 post-infection (pi). CD8a-d2EGFP is relatively stable and remains expressed even in the quiescent, latently infected cells.

Article Snippet: Continued REAGENT or RESOURCE SOURCE IDENTIFIER IL-7 PeproTech 200-07 IL-8 PeproTech 200-08M 1L-10 PeproTech 200-10 IL-23 PeproTech 200-23 TGF-b PeproTech 100-21C Anti-human IL-4 PeproTech 500-M04 Anti-human IFN-g PeproTech 500-M90 Concanavalin A MilliporeSigma C0412 Critical commercial assays Human CD4 naive T cell isolation kit BioLegend 480042 Mouse CD8a positive selection kit STEMCELL 18953 Pacific Blue antibody labeling kit Thermo Fisher Scientific P30013 Alexa Fluor 488 antibody labeling kit Thermo Fisher Scientific A20181 Alexa Fluor 555 antibody labeling kit Thermo Fisher Scientific A20187 Alexa Fluor 647 antibody labeling kit Thermo Fisher Scientific A20186 Experimental models: cell lines HEK293T ATCC CRL-3216 Experimental models: Organisms/strains Polarized and expanded primary Th17 cells This paper N/A Recombinant DNA pHR’-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 pHR’-CD8a-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 Lentiviral gag/pol pCMV-Delta 8.91r Dobrowolski et al.2 Dobrowolski et al.2 pMD.G VSVG Addgene #8454 Other Optima L-100K ultracentrifuge Beckman Coulter N/A SW32 Ti rotor Beckman Coulter N/A BD LSR Fortessa BD Biosciences N/A EasySep magnetic tube holder STEMCELL 18000 Eppendorf 5810R benchtop centrifuge Eppendorf 022625101 STAR Protocols 5, 103398, December 20, 2024 3 Day 2: Generation of single-round VSVG-pseudotyped HIV 2.

Techniques: Infection, Virus, Construct, Selection

Journal:

Article Title: Human Immunodeficiency Virus Type 1-Derived Lentivirus Vectors Pseudotyped with Envelope Glycoproteins Derived from Ross River Virus and Semliki Forest Virus

doi: 10.1128/JVI.78.3.1421-1430.2004

Figure Lengend Snippet: Envelope glycoprotein expression plasmids. (A) The original VSV-G expressing plasmid (pMD.G) contains both the rabbit β-globin intron and poly(A) signal. The expression plasmids for RRV and SFV glycoproteins (pRRV-E2E1A and pSFV-E2E1A, respectively) do not have an intron and also have a different poly(A) signal. (B) Standardized glycoprotein expression plasmids were constructed that have both the rabbit β-globin intron and the rabbit β-globin poly(A) signal from pMD.G to standardize expression of VSV, RRV, and SFV glycoprotein expression cassettes.

Article Snippet: Then, the VSV(Indiana)-G expressing plasmid pMD.G (Cell Genesys, Foster City, Calif.) ( 34 ) was digested with SnaB I and Xba I, and the fragment containing the VSV-G coding region, together with the rabbit β-globin intron and its poly(A) signal, was ligated into the Sna BI and Xba I sites in the pCI-DestB expression vector.

Techniques: Expressing, Plasmid Preparation, Construct