Lenti x p24 rapid titer kit

The Lenti-X p24 Rapid Titer Kit from TaKaRa was used following the manufacturer’s protocol. Further details are provided in S1 Methods.

The p24 content of HIV-infected cell culture supernatants was measured with the Lenti-X™ p24 Rapid Titer Kit from Takara Bio (Mountainview, CA). Supernatants from each control condition representing the highest expected virus content were removed (5 µL) and diluted to empirically determine the optimal dilution factor for a given experiment to enable measurements in the linear range of the assay (12.5–200 pg/mL), which typically required 10³- to 10⁴-fold dilutions. Absorbance was measured at 450 nm. Values are recorded as the concentration and percentages of an untreated control.

Production of lentiviral particles pseudotyped with MERS S glycoprotein was performed as previously described [17 (link)]. Briefly, the human immunodeficiency virus backbone expressing firefly luciferase (pNL4.3R-E-luciferase) were co-transfected into 293T cells, either with the MERS-CoV wild-type spike glycoprotein (Wt S) expression vector (pcDNA3.1+; Invitrogen) or with the mutant D510A spike glycoprotein vector which significantly reduces its binding with DPP4 and consequently the viral entry [17 (link)] (Figure 1A). Supernatants containing the lentiviral particles pseudotyped with MERS-CoV S glycoprotein were harvested 48 h later and were subsequently normalized using a p24 ELISA kit prior to infection of the target cells (Lenti-X™ p24 Rapid Titer Kit, Takara Bio USA, Inc.). The amount of Spike glycoprotein incorporated in the different types of the generated viral particles pseudotyped with the MERS-CoV S protein was determined by semi-quantitative Western blot in purified viral particles, ensuring an equal MERS-CoV S glycoprotein expression among them. Infected cells were lysed at 48 h after infection and viral entry efficiency was quantified by comparing the luciferase activity between the pseudotyped viruses either not expressing the MERS-CoV S glycoprotein or bearing the mutant and wild-type MERS-CoV S glycoprotein.

Approximately 750,000 low-passage 293FT cells were seeded into each well of a 6-well plate coated with poly-L-ornithine (Sigma-Aldrich, P2533). One day after seeding, cells were transfected with 640 ng pMD2.G, 975 ng psPAX and 1275 ng lentiviral target plasmid using Lipofectamine 2000 and PLUS reagent (Invitrogen, 11514015). The medium was refreshed 4 h after transfection. The supernatant was collected 48 and 72 h post transfection, filtered with a 0.45 μm filter (Millex-HV, SLHV013SL), and concentrated with Lenti-X Concentrator (Takara Bio, 631231) according to the manufacturer's instructions. Physical titer was determined using the Lenti-X p24 Rapid Titer Kit (Takara Bio, 632200) and granule cell precursors were transduced at an estimated multiplicity of infection of ∼4.

LV titers were measured using the Lenti-X™ p24 Rapid Titer Kit (Takara Bio USA, Inc.). Reverse transcriptase (RT) activity was measured using the EnzChek^® Reverse Transcriptase Assay (ThermoFisher Scientific). Post-production of particles, HEK 293T (for LVs) or HEK 293 (for JSRV, ENTV and the chimeras) cells were lysed, or their supernatant was harvested for ultracentrifugation prior to lysing with RIPA buffer and processed for western blot or EM. Protein expression was visualized via WB and vector particles were imaged using EM, as described previously [31 (link)]. JSRV, ENTV and the chimeras were titered via RT assay and p27 ELISA. Tissue slices were infected with 1 × 10⁶ infectious units (IFU), as previously described [27 (link)].

Latency reversal and HIV-1 production were measured by performing p24-ELISA with the culture supernatants using the Lenti-X p24 Rapid Titer Kit (TaKaRa). Briefly, the culture supernatant from the edited and control samples was diluted 1:40-fold in the complete DMEM medium used for ELISA as per the manufacturer’s instructions. The OD₄₅₀ value for the blank control was subtracted from the value of each sample. Three technical replicates for each of the six biological replicates were averaged, and standard deviation was calculated. The fold change in supernatant p24 (proxy for HIV production) and P value were calculated by comparison with the NTC of the same donor.