A Protocol for Studying HIV-1 Envelope Glycoprotein Function

Summary HIV-1 envelope glycoproteins (Envs) bind to CD4 receptor and CCR5/CXCR4 coreceptor and mediate viral entry (Feng et al., 1996; Herschhorn et al., 2016, 2017; Kwong et al., 1998). HIV-1 Envs are the sole target of neutralizing antibodies and a main focus of vaccine development (Flemming et al., 2018). Here, we provide a step-by-step protocol to measure Env sensitivity to ligands, cold, and small molecules, as well as to study viral infectivity and to dissect parameters affecting HIV-1 Env function. For complete details on the use and execution of this protocol, please refer to Harris et al. (2020).

cells for measuring PV infectivity. Cells can be stored for years in the vapor phase of a liquid nitrogen storage tank.
Note: All cells should be passaged at least twice and should show healthy morphology before using them in the viral assays. We have not seen significant decrease in transfection efficiency (293T) or reporter activity (TZM-bl or Cf2Th-CD4/CCR5) even after 30 passages. We typically split the cells at <90% confluency (every 2-3 days) using the following protocol: (1) Remove the media from the flask, followed by washing once with PBS.
(3) Add 10 mL of DMEM to the flask, collect the detached cells, and mix slowly by gentle pipetting up and down. (4) Dilute the cell suspension as necessary and transfer the required volume of cells to a new flask. All cells are typically maintained in vented culture flasks at 37 C, incubated with 5% CO 2 .
Alternatives: Mix & Go E. coli transformation kit (T3001; Zymo Research) can be used to avoid the need for heat shock, incubations, or outgrowth steps.
7. Select from the transformed bacteria plate a well separated and rounded single bacteria colony. 8. Inoculate the bacteria in 2 mL Luria Bertani (LB) broth supplemented with 100 mg/mL ampicillin or carbenicillin (a stable derivative of ampicillin). ll OPEN ACCESS 9. Grow the transformed bacteria for 16 h in a shaker incubator at 37 C for small scale minipreps or dilute them after 6-8 h to 250 mL of the same LB medium containing antibiotics and allow the bacteria to grow overnight (16-20 h) for medium scale midipreps.
Note: Several commercial miniprep and midiprep kits are available from different vendors to purify plasmid DNA. Follow the manufacturer's instructions.
CRITICAL: Transformed pHIVec2.luc plasmid is maintained in E. Coli bacteria at a low-copy number and therefore the pHIVec2.luc plasmid should be prepared from a large volume culture. We usually grow the transformed bacteria in two to three 1-liter flasks containing bacteria in 250 mL LB broth each. Split the 23 HEPES buffer to three beakers, 35 mL in each, and titrate the three solutions to pH 6.8, pH 7.05, and pH 7.2 using 5M NaOH. Add ddH 2 O up to 40 mL to each beaker, filter-sterilize the three solutions using 0.22 mm syringe filter, aliquot in 1.5 mL microcentrifuge tubes and store at -20 C.

KEY RESOURCES
Prepare 2M calcium chloride in ddH 2 O solution. Aliquot the solution in 1.5 mL microcentrifuge tubes and store at -20 C.
Alternatives: These solutions can be purchased commercially as a calcium phosphate transfection kit (e.g., K278001 from Invitrogen).
Note: pH is critical for transfection; all three buffers at different pH values should be tested by transfection of a plasmid containing reporter gene (e.g., green fluorescent protein (gfp)) and measuring the expression level of the reporter protein or the efficiency of transfection. We routinely achieve >90% transfection efficiency with plasmid containing the gfp gene and optimal Ca 3 (PO 4 ) 2 reagents. Solutions are usually stable for only 1-2 months at -20 C.   CRITICAL: H 2 SO 4 is a highly corrosive chemical and should be handled in a chemical hood with gloves, eye glasses, and proper PPE.
Titrate with 15% Phosphoric acid (H 3 PO 4 ) to pH 7.8 and add ddH 2 O up to 200 mL. Store at 4 o C.

Luciferin Substrate Solution
Reconstitute lyophilized Luciferin with ddH 2 O and adjust the pH to 6.0-6.3 with HCl/NaOH. Store in aliquots at -20 C. Work quickly and protect from light for any luciferin-based solutions.
Alternatives: Luciferase assay system that does not require the use of a luminometer with injectors can be purchased commercially (e.g., Bright-Gloä Luciferase Assay System from Promega).

Spectrophotometer
A spectrophotometer microplate reader is required to measure optical density at the last step of the p24 ELISA in order to measure the concentration of HIV-1 p24 in PV preparations.

Vacuum System
We installed an in-house vacuum system connected through central vacuum supply (Figure 2). In this setup, the central vacuum line is sequentially connected to a 0.22 mm filter and two collection flasks (traps) followed by an aspirating tube. A serological pipette is connected to the tube for focused aspirating. This vacuum system provides manual flow control and works efficiently for aspirating the liquid from cell culture plates during ELISA procedures or before adding the lysis buffer to 96well assay plates.

Luminometer
A luminometer is required to measure the reporter luciferase activity, which results in an extremely sensitive readout with a dynamic range of more than 7 orders of magnitude. Since luciferin readout decays exponentially with a very short half-life, a luminometer equipped with injectors is required to read each well after a predefined time window to ensure reproducible results. We use the Centro XS 3

Reagent
Final Concentration Stock Concentration Amount Volume

STEP-BY-STEP METHOD DETAILS Production of Recombinant HIV-1 Pseudoviruses
Timing: 1 week 1. Day 0: Maintain 293T cells in DMEM medium and monitor their growth rate and morphology. Cells should grow exponentially with a doubling time of approximately 24 h and show healthy morphology without significant cell aggregation. Wash 293T cells once with PBS and add 1-2 mL of StemPro Accutase, which gently detaches the 293T cells while preserving the expression of the receptors on the cell surface. Incubate at RT (20 C-25 C) until cells are completely detached, add 10 mL of DMEM to the flask, collect the detached 293T cells, and mix slowly by pipetting up and down. The cell suspension can be filtered if they tend to aggregate. We usually use 5 mL polystyrene round-bottom tube with cell-strainer cap to filter the cell suspension. 2. Count the cells using a hemocytometer and add 5 3 10 5 cells/well to a 6-well plate or 3 3 10 6 cells/T-25 flask. Incubate the plate/flask overnight (14-20 h) in the tissue culture incubator at 37 C and 5% CO 2 concentration. 3. Day 1: Effectene transfection.
a. Replace the medium with a fresh DMEM medium and follow manufacturer's instructions. A typical template for preparing the transfection mixture for a 6-well plate transfection is shown in Table 1. Add dropwise the transfection mixture to the cells with gentle swirling.
b. Gently swirl the plate/flask to ensure uniform distribution of the transfection complex. Incubate plate/flask in a tissue culture incubator at 37 C and 5% CO 2 concentration. 4. Alternatives: Ca 3 (PO 4 ) 2 transfection. a. Prepare 293T cells for transfection as described in day 0 but incubate the plate/flask for only 4-5 h in tissue culture incubator at 37 C and 5% CO 2 concentration to allow the cells to attach to the surface. b. Add chloroquine to a final concentration of 25mM, incubate for 5 min, and prepare the Ca 3 (PO4) 2 transfection mixture. A typical template for preparing the transfection mixture for a T-25 flask transfection is shown in Table 2. Add dropwise the transfection mixture to the cells with gentle swirling. Note: We found that Ca 3 (PO4) 2 transfection of 293T cells is more efficient 4-5 h after seeding the cells in comparison with 293T cells that were grown overnight (14-20 h) prior to transfection.
Note: According to our institutional biosafety approved protocol, at this point we transfer the plate/flask to our BSL2+ facility. Work at the BSL2+ facility follows BSL3 practices in a BSL-2 environment. The standard personal protective equipment includes disposable gown, face mask, protective shatterproof eyeglasses or face shield, double gloves, sleeve cover, and protective shoe covers. Liquid waste is decontaminated with 10% bleach and all waste is autoclaved on site.
CRITICAL: Chloroquine is toxic to cells; medium must be changed 14-16 h post transfection.
6. Day 3: Gently collect the PV-containing supernatant and centrifuge at 600-900 3 g for 5 min at 4 C to remove cell debris. Collect the supernatant and aliquot in required volume for single-use experiments in several 1.5 mL microcentrifuge tubes. One tube should be reserved for a p24 measurement (next section).
Alternatively, PV-containing supernatant can be filtered through a 0.45 mm filter. The two alternatives (centrifugation and filtration) may be compared for specific Envs to achieve high infectivity. Some studies have used a centrifugation step followed by 0.45 mm filtration of the PV-containing supernatant.
7. Store PV-containing tubes at À80 C for future use. PV infectivity depends on the specific HIV-1 Env used and is typically decreased $90% with a freeze-thaw cycle. Thus, viruses pseudotyped with Envs that intrinsically exhibit very low entry activity cannot be frozen and have to be used immediately.
CRITICAL: PVs should be collected with minimal mixing or minimal use of any mechanical force to preserve virus infectivity.
8. After collecting the supernatant, lyse the remaining cells in the wells/flask with luciferase lysis buffer. Transfer a few microliters of lysed cells (usually 1:10 or 1:100 of the lysate) to a 96-well white luminometer plate and measure luciferase activity. The readout reflects the efficiency of pHIVec2.luc transfection and should be close to saturation. We typically measure >2 x 10 9 relative light unit for 2-s measurements for such cell lysates.
Note: If the transfection efficiency is high, the cell lysate readout may be saturated even at 1:100 dilution. Transfection efficiency determines the relative PV titer and p24 concentration in the sample. 9. Day 3 (or 4): Each HIV-1 virion (particle) contains approximately 3000 copies of p24 protein. Thus, after viruses are disrupted to release the p24 protein, the HIV-1 p24 concentration in a specific preparation is related to the number of virions in the sample. The concentration of HIV-1 p24 protein can be measured using commercial or in-house enzyme linked immunosorbent assay (ELISA) based on HIV-1 p24 antigen capture. An efficient and economical in-house assay has been previously described (Wehrly and Chesebro, 1997); commercial p24 ELISA kits (e.g., ABL p24 capture assay) are usually more expensive, are provided in adjustable 8-well strips, and typically detect p24 concentrations at the range between 5 and 100 pg/mL in a test sample. Here we describe the use of the duo ELISA set from R&D, which provides only main components of the p24 ELISA system and represents a method of high performance at an attractive price. Using this set, we reproducibly measure p24 concentrations within a range of 31 to 500 pg/mL. 10. Duo p24 ELISA protocol: a. Add equal volume of PV sample to a tube containing lysis buffer (1% Triton X-100 in PBS). The lysed sample can be stored at À20 C until use. b. Immobilize a capture antibody (HIV Gag p24 Capture Antibody, Part 844721; Cat# DY7360-05; R&D systems) on high-binding 96-well plate (Greiner bio-one, Cat# 655061) by adding 0.4 mg of antibody in 100 mL PBS (4 mg/mL) to each well. Seal the plate and incubate overnight (14-20 h) at RT (20 C-25 C). c. The following day wash the wells as follows.

Wash
Step: aspirate the solution from each well using a vacuum system and wash three times with 300 mL of wash buffer by adding the buffer, incubating for 30 s, and aspirating the solution. Invert the plate on a paper towel to remove remaining traces of wash buffer. d. Add 300 mL of blocking buffer to each well and incubate the plate at RT (20 C-25 C) for 1-2 h. Repeat Wash Step. e. Add 100 mL/well of p24 protein standards (HIV Gag p24 Standard, Part 844723; Cat# DY7360-05; R&D systems) at the following concentrations (serial 1:2 dilutions): 500, 250, 125, 62. 5, 31. 25, 15.625, 7.8125, and 0 (blank) pg/mL in duplicates. The measurements of these wells will be used to generate a standard curve. In addition, add 100 mL/well of test samples to respective wells. We typically test 2-3 dilutions of each sample to ensure that the measurements are within the range of the standard curve. Incubate the plate at RT (20 C-25 C) for 2 h and then repeat the Wash Step. f. Add 100 mL/well of detection antibody (HIV-1 Gag p24 Detection Antibody, Part 844722; Cat# DY7360-05; R&D systems) at a final concentration of 125 ng/mL and incubate at RT (20 C-25 C) for 1 h. At the end of incubation repeat the Wash Step. g. Add 100 mL/well of 1:40 dilution of streptavidin-HRP solution (Part 893975; Cat# DY7360-05; R&D systems) and incubate the plate at RT (20 C-25 C) for 20-30 min. At the end of incubation repeat the Wash Step. h. Add 100 mL/well of TMB substrate solution and incubate at RT (20 C-25 C) for 15-20 min followed by the addition of 50 mL/well stop solution. Shaking will improve the distribution of color development in the well. Within 5-30 min, measure the optical density in each well at wavelength of 450 nm. Protect the plate from light during this step. We usually cover the plate with an aluminum foil immediately after the addition of the TMB substrate.

OPEN ACCESS
i. Analyze the data. Measurements are averaged and the 0 (blank) readout is subtracted from each. Averaged and blank-subtracted p24 measurements are then plotted against each p24 concentration and a standard curve is generated by fitting the measurements to a linear curve using available processing program (e.g., Excel, GraphPad). p24 concentration in a tested sample is calculated using the equation describing the fitted linear curve. R 2 values are typically > 0.97.
CRITICAL: According to our institutional biosafety approved protocol, once the PV is lysed we transfer the plate/flask to a BSL2 laboratory. Liquid waste is decontaminated by 10% bleach for 20 min.
Note: Another way to compare PVs is to measure the level of Env expression on the surface of each PV, which can be done by analyzing the proteins in PV preparation by western blot.

Timing: 3-4 days
To compare the properties of different Envs or different preparation of the same Envs, equivalent amounts of PVs are used according to (1) number of virions, which is estimated by p24 concentrations of the preparation (as described in the previous section), or (2) viral infectivity levels of Env-displaying PVs. We next describe how to titer PVs on target cells; the infectivity values measured can be used to generate a dose-response curve and to estimate the volume of each PV preparation that is required for desired infectivity level.
11. Day 1: Prepare serial 2-fold dilutions of supernatant-containing PVs in DMEM (i.e., 1:2, 1:4, 1:8, .). PVs are very sensitive to mechanical forces and mixing should be gentle and minimal to allow even distribution of PVs in the solution without decreasing viral infectivity. 12. Add 30 mL/well of diluted PVs in duplicates or triplicates to a 96-well plate. We compared several 96-well plates and, in our hands, Greiner bio-one 96-well plates (Cat# 655083) give low background and high signal. We usually use internal wells for viral assay and add 120 mL of DMEM to otherwise empty wells to prevent evaporation of liquid in the assay wells (edge effect). An example of a typical template is shown in Figure 3. 13. Add approximately 0.5 mL of StemPro Accutase to a T75 flask containing Cf2Th-CD4/CCR5 target cells and incubate at RT (20 C-25 C) until cells are completely detached. Target cells should be grown no more than 2 days prior to the assay to ensure healthy and exponentially growing cells. StemPro Accutase is a gentle detachment agent and preserves the receptor integrity on the cell surface. Add 10 mL DMEM, centrifuge the cells at 50-100 3 g and 4 C for 5 min, remove the supernatant, and resuspend the target cells in DMEM at concentration of 1.2 3 10 5 cells/mL. 14. Add 60 mL/well of cells (7200 cells/well) to the wells containing PVs from step 12. Control wells should include: target cells with no PVs and PVs with no cells in the same total volume as all other wells (90 mL/well). Incubate the plate in a tissue culture incubator at 37 C and 5% CO 2 concentration.
Alternatives: Target cells can be added to a 96-well plate (7200 cells/well) one day prior to the viral assay followed by the addition of viruses on the next day.
CRITICAL: We found that adding target cells immediately after PV addition results in significantly higher assay readout compared with seeding target cells one day prior to assay.
Note: Infection of Cf2Th-CD4/CCR5 target cells with PVs typically results in higher assay readout than the readout of infection of TZM-bl target cells. But TZM-bl cells have been extensively used in numerous reported studies. These cells contain a stably integrated copy of the firefly luciferase gene under the control of HIV long terminal repeat promoter in their genome, and exhibits relatively high background. TZM-bl cells are contaminated with ecotropic murine leukemia virus but this contamination does not significantly affect their performance as target cells (Platt et al., 2009). The addition of DEAE-Dextran increases the efficiency of viral entry.
15. Day 3 (or day 4): PV entry into target cells can be measured after 48 or 72 h. Measurements 72 h post infection usually results in higher readout than the measurements after 48 h, most probably because the firefly luciferase enzyme is accumulating in the target cells during this time. However, 48-h incubation typically results in high signal-to-noise ratio and in many cases is preferred due to time saving. a. Prepare sufficient luciferase assay buffer to measure the luciferase activity in the assay wells (100 mL/well). Thaw the luciferin substrate solution at RT (20 C-25 C); thawing can be accelerated by immersing the tube in a beaker filled with water at RT (20 C-25 C). b. Gently aspirate the medium from the wells of the 96-well assay plate using a vacuum system. Add 30 mL/well of luciferase lysis buffer to lyse the cells and measure the activity of luciferase using a luminometer plate reader equipped with two injectors. In our hands, Berthold luminometer provides robust and reproducible readings. After priming the injectors with the assay buffer (injector 1) and the luciferin substrate solution (injector 2), one hundred microliters of assay buffer are injected to each well, followed by a 1-s delay and a 50-mL injection of luciferin substrate solution. Light units are measured and integrated over between 2 and 10 s.
CRITICAL: Luciferin solution is light sensitive; keep solution protected from light at all times.
Note: All luciferase reagents and the sample plate should be allowed to equilibrate to RT (20 C-25 C) before measuring luminescence as temperature variations could affect the assay readout.

Effects of HIV-1 Env Ligands on PV Infectivity
Timing: 3-4 days ll

OPEN ACCESS
To measure the effect of ligands on specific Env function, PVs displaying specific Envs are incubated with increasing concentrations of the tested ligand followed by the addition of target cells to each well to measure infectivity (Herschhorn et al., 2014). Wells of infected cells without a ligand are used as reference control (100% infection) and wells with cells only are used as background control (0% infection). Additional control wells contain PV without cells. The readout for each concentration of the ligand is normalized to the two control values.
16. Day 1: Serially dilute the tested ligand in DMEM. Calculate the amount and volume needed for duplicate or triplicate measurements and dilute the ligand in microcentrifuge tubes. For small molecules dissolved in DMSO, dilute the compounds in DMSO and then dilute further to final concentration in DMEM while keeping identical DMSO concentration for all wells. Up to 2% DMSO can be used in the viral assay without any cytotoxic effects to Cf2Th-CD4/CCR5 target cells. DMSO must be included in the reference infection. An example of template is shown in Figure 4.
17. Add 30 mL of ligand at specific concentration to wells of a 96-well luminometer plate (Greiner bio-one 96-well plate (Cat# 655083)). Each ligand concentration is usually tested in duplicates or triplicates. Use 30 mL of the diluting medium for reference control (no ligands) and background (cells only) wells. 18. Add 30 mL PVs that were prediluted either to a defined amount of HIV-1 p24 or to specific infection level (or titer) to assay and reference control wells. We typically use a specific HIV-1 p24 amount in the range of 1-10 ng or infectivity values equivalent to about 1 million relative light units. 19. Repeat step 13 of Virus titration section but resuspend the target cells in DMEM at concentration of 2.4 3 10 5 cells/mL. Add 30 mL/well of cells to the wells containing PVs, which were preincubated with a ligand, and to reference and background control wells. 20. Add 30 mL of medium to background control wells. Incubate the plate in a tissue culture incubator at 37 C and 5% CO 2 concentration. 21. Day 3 (or day 4): Repeat step 15 (a & b) of the previous section (Virus titration).
Note: Normalizing by p24 concentration reflects equivalent number of viral particles in a sample. Normalizing by PV infectivity reflects the number of viral particles that are able to mediate similar levels of entry into target cells.
Note: Measuring ligands dissolved in DMSO requires the addition of DMSO to the reference cells to account for the DMSO effect

Cold Sensitivity
Timing: 5-6 days We and others have previously shown that frequent sampling of the open conformation of HIV-1 Env is associated, in many cases, with increased Env sensitivity to exposure to cold. This Env property is studied by exposing PVs to low temperature (ice) at defined intervals followed by measuring the effect on infectivity. Because different PV-containing tubes of the same batch are thawed at different time points and kept on ice for defined time frames, care should be taken to maintain PV infectivity in each tube during virus handling.
22. Day 1: Prepare approximately 10 mL of PV displaying specific HIV-1 Envs according to the steps outlined in the first section (Production of recombinant HIV-1 PVs). Aliquot the supernatant-containing PVs into 20 or more microcentrifuge tubes and store at À80 C. Measure HIV-1 p24 in the supernatant and titer the PVs as described in the previous section (Virus titration). 23. Thaw a single-use aliquot of PV preparation by immersing the tube in a 37 C water bath for exactly 1.5 min and then gently place the tube in an ice bucket. Place the ice bucket in a refrigerator to conserve the ice as much as possible during incubation. Label the tube as day 1. 24. Repeat step 23 daily for 3 consecutive days (days 2-4) at the same interval (approximately every 24 h). On day 5, repeat step 23 but without placing the tube on ice. PVs from day 5 tube are the reference for measuring 100% infectivity. 25. Dilute each PV preparation to 4 ng p24/60 mL or to a predefined infectivity level in DMEM. Add 60 mL of each PV in triplicates or quadruplicates to wells of a 96-well plate followed by the addition of 60 mL of 1.2 3 10 5 Cf2Th-CD4/CCR5 target cells/mL (7200 cells/well). Incubate the plate in a tissue culture incubator at 37 C and 5% CO 2 concentration. 26. Day 3 (or day 4): Repeat step 15 (a & b) of the previous section (Virus titration).

Cell-Cell Fusion Assay
Timing: 3-4 days Cell-cell fusion assay measures the fusion activity of specific HIV-1 Envs that are tested (Herschhorn et al., 2011). Effector cells are transfected with HIV-1 Tat-and HIV-1 Env-expression plasmids. TZMbl cells serve as target cells. They express the CD4/CCR5 receptors and contain a stably integrated copy of the firefly luciferase gene under the control of HIV long terminal repeat promoter in their genome. Fusion of effector and target cells during co-culture leads to diffusion of HIV-1 Tat to the target cells and activation of firefly luciferase transcription. 27. Day 1: Follow the steps outlined in the first section for Ca 3 (PO 4 ) 2 transfection (step 4) to transfect 293T cells with HIV-1 tat-and HIV-1 Env-expression plasmids at a ratio of 1:6 in a 6-well plate (Table 3). Incubate transfected cells in a tissue culture incubator at 37 C and 5% CO 2 concentration.
28. Day 2: Detach TZM-bl cells and centrifuge the cells at 50-100 3 g and 4 C for 5 min. Resuspend the cells in DMEM, count the cells and adjust the concentration of 1 3 10 5 cells/mL. Add 100 mL of cells to designated wells of a 96-well plate ( Figure 5). 29. In addition to assay wells containing co-culture of effector (293T) and target (TZM-bl) cells, wells containing only effector cells or only target cells are used as control. We usually measure fusion after 2, 4, and 6 h; each time point requires a separate plate. See an example layout in Figure 5. Incubate the plate overnight (16-20 h) in a tissue culture incubator at 37 C and 5% CO 2 concentration. 30. Day 3: Detach the transfected 293T cells using 5 mM EDTA in PBS and transfer to a 15-mL centrifuge tube. Add 10 mL DMEM and centrifuge cells at 50-100 3 g and 10 C for 5 min. Resuspend the transfected 293T cells in fresh medium at concentration of 2 3 10 5 cells/mL. 31. Remove 50 mL from assay wells that are designated for co-culture and contain TZM-bl target cells, and add 50 mL of transfected 293T cells (10,000 cells/well). Repeat this step for empty wells that contain DMEM medium and are designated as control. Keep wells with TZM-bl cells only as second control ( Figure 5).
32. Incubate the plates in a tissue culture incubator at 37 C and 5% CO 2 concentration and measure the fusion activity after 2, 4, and 6 h (1 plate for each time point) by following step 15 (a & b) of the Virus titration section.

EXPECTED OUTCOMES
Our protocol provides molecular tools to study the function of HIV Envs. We usually produce high titer PVs from one T-25 flask or 6-well plate of transfected 293T cells (3 million cells/flask; 0.5 million/well of 6-well plate) using both Ca 3 (PO 4 ) 2 as well as commercial Effectene transfection reagent. Based on luciferase expression system, signal-to-noise ratio for highly efficient HIV-1 Envs (e.g., HIV-1 JRFL Envs, see below) should be > 1000. The experiments should give the following information for each specific Envs: 1. Sensitivity to ligands (e.g., antibodies, sCD4). This is usually calculated as IC 50 (see below). 2. Fusion ability. Comparison of the fusion activity between different Envs. 3. Sensitivity to cold. Envs of many primary HIV-1 strains are not sensitive to cold exposure up to at least 96 h. This property usually correlates with a more closed conformation. Nevertheless, some Integration of the above Env properties will provide a comprehensive view on HIV-1 Env function and a basis for comparison between Envs of different strains as well as a framework to study the effect of amino acid changes on HIV-1 Env activity.

QUANTIFICATION AND STATISTICAL ANALYSIS
The detailed example in Figure 6 provides a step-by-step guidance for calculating half maximal inhibition concentration (IC 50 ) of HIV JRFL Envs by soluble CD4. The assay was performed in triplicates in a 96-well plate according to our protocol. Results are adapted from Harris et al. Cell Reports 2020. Figure 6. A Workflow for IC 50 Calculation A step-by-step analysis of dose-response data generated measuring the effect of different concentrations of sCD4 on HIV-1 JRFL pseudovirus infection. We typically calculate the average values using an Excel spreadsheet and then export the data to Prism (GraphPad). We fit the data to the logistic equation after adding the equation to the program (Herschhorn et al., 2008(Herschhorn et al., , 2010. For typical dose-response curves, the following constrains are used for consistency: max value = 100%; min value = 0%; p < 3. Ligands that either enhance infection or do not completely inhibit infections should be treated individually and all parameters (Max, Min, IC 50 , and p) should be reported.

LIMITATIONS
Our protocol cannot measure HIV-1 Env function of Envs with very low fusion activity. Some primary HIV-1 strains can replicate in cells but they enter target cells with very low efficiency. As a result, we will not be able to detect the viral entry mediated by of some Envs in our system. For example, in our hands a codon-optimized version of BG505 showed a very low infectivity and could not been used for accurate and reliable measurements. Other Envs do not tolerate freeze-thaw cycle and have to be assayed immediately after PV preparation. In this case, p24 is measured immediately after PV preparation and the virus is then used. Alternatively, several dilutions can be used for the viral assay immediately after preparation, and p24 concentration of each dilution is determined at a later time point. The latter approach does not allow to use predefined p24 concentration but a dilution with desired p24 concentration can be selected for analysis. PV infection follows Poisson distribution and low levels of infection usually result in high variability between the readout from different wells on the same plate and between the measurements of independent experiments. Thus, reliable results require Envs that can mediate reasonable level of infection or can be concentrated without significant loss of activity. PVs can be concentrated by ultracentrifugation, membrane-based anion exchange chromatography or precipitation using polyethylene glycol 6000 (Kutner et al., 2009;Reiser, 2000).

Potential Solution
Test the luciferase assay reagents: luciferin substrate, assay buffer, DTT and ATP; and prepare new reagents if needed. Make sure 293T cells are healthy. If needed, thaw a new tube of 293T cells. Test transfection reagents with a reporter protein Prepare PVs with efficient Envs (e.g., HIV-1 JRFL Envs) to rule out technical problems during PV preparations Use PVs without freezing. Envs of some HIV-1 strains are poor and sensitive to freeze-thaw cycles. The polycation DEAE-Dextran can be supplemented in the assay medium to increase the PV infectivity while performing TZM-bl neutralization assays (https://www.hiv.lanl.gov/content/nab-reference-strains/ html/Protocol-for-Preparation-and-Titration-of-HIV-1-Env-pseudotyped-Viruses_Apr-2020.pdf). However, including DEAE-Dextran in the assay can result in up to 3-fold change in the neutralizing antibody activity (Sarzotti-Kelsoe, M et al., 2014). Thus, if DEAE-Dextran is used in experiments that compare different viruses it should be used for all samples tested.

Potential Solution
Use higher titer of PV. In our hands, infection of Cf2-CD4/CCR5 cells for 48 h that leads to approximately 1 3 10 6 relative light units, which are measured during 2-s integration on the Centro LB 960 XS 3 Berthold luminometer, gives reliable and reproducible readouts. Incubate PV-infected target cells for 72 h. Readout are usually higher and more uniform after a 72h incubation Make sure Luciferin substrate is at RT (20 C-25 C) during the assay measurements

Problem 3
Cell aggregates when preparing 293T cells for cell-cell fusion (step 27).

Potential Solution
Gently mix with serological pipette to break cell aggregates Filter the cells and then count cell number

RESOURCE AVAILABILITY
Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Alon Herschhorn (aherschh@umn.edu).

Materials Availability
Materials generated in this study are available upon request.

Data and Code Availability
This study did not generate any unique datasets or code.