Identification of CD38, CD97, and CD278 on the HIV surface using a novel flow virometry screening assay

While numerous cellular proteins in the HIV envelope are known to alter virus infection, methodology to rapidly phenotype the virion surface in a high throughput, single virion manner is lacking. Thus, many human proteins may exist on the virion surface that remain undescribed. Herein, we developed a novel flow virometry screening assay to discover new proteins on the surface of HIV particles. By screening a CD4+ T cell line and its progeny virions, along with four HIV isolates produced in primary cells, we discovered 59 new candidate proteins in the HIV envelope that were consistently detected across diverse HIV isolates. Among these discoveries, CD38, CD97, and CD278 were consistently present at high levels on virions when using orthogonal techniques to corroborate flow virometry results. This study yields new discoveries about virus biology and demonstrates the utility and feasibility of a novel flow virometry assay to phenotype individual virions.


CD38 CD44
Preanalytical variables relating to EV sample including source, collection, isolation, storage, and any others relevant and available in the performed study.
Virus containing cell supernatants were generated, collected, and stored as described in the Materials and Methods section.No further isolation techniques were used.
1.2 Experimental design according to MIFlowCyt guidelines.
EV-FC manuscripts should provide a brief description of the experimental aim, keywords, and variables for the performed FC experiment(s) using MIFlowCyt checklist criteria: 1.1, 1.2, and 1.3, respectively.Template found at www.evflowcytometry.org.

Sample staining details
State any steps relating to the staining of samples.Along with the method used for staining, provide relevant reagent descriptions as listed in MIFlowCyt guidelines (Section 2.4 Fluorescence Reagent(s) Descriptions).
Virus samples were stained with PE conjugated antibodies against cellular proteins.Methods used for staining are as described in in the Materials and Methods.

Sample washing details
State any steps relating to the washing of samples.Virus samples were not washed.

Sample dilution details
All methods and steps relating to sample dilution.
Unlabelled virus supernatants were serially diluted prior to acquisition on the flow cytometer to estimate virus particle concentration.Antibody labeled virus samples were diluted 1:250-1:1000 with PBS prior to acquisition on the flow cytometer.See methods in the manuscript for detailed description.
State whether a buffer-only control was analyzed at the same settings and during the same experiment as the samples of interest.If utilized it is recommended that all samples be recorded for a consistent set period of time e.g. 5 minutes, rather than stopping analysis at a set recorded event count e.g.100,000 events.This allows comparisons of total particle counts between controls and samples.
Media only sample controls were run (Fig. S3).
3.2 Buffer with reagent controls.
State whether a buffer with reagent control was analyzed at the same settings, same concentrations, and during the same experiment as the samples of interest.If used state what the results were.
Media containing antibody controls were run for each individual antibody used in Figure 5.

Unstained controls.
State whether unstained control samples were analyzed at the same settings and during the same experiment as stained samples.If used, state what the results were, preferably in standard units.
Unstained virus samples were analysed using the same settings as stained samples (Fig. S3).
3.4 Isotype controls.The trigger channel(s) and threshold(s) used for event detection.Preferably, the fluorescence calibration (Section 4.3) and/or scatter calibration (Section 4.4) should be used in order to report the trigger channel(s) and threshold(s) in standardized units.
Trigger Channel(s) and Threshold(s) in calibrated and arbitrary units are summarized for each sample in the table on the 'Sample Acq Sheet'.
State if the flow rate was quantified/validated and if so, report the result and how they were obtained.
Flow Rate Volumetric quantification was performed using the application in the Beckman Coulter CytExperts acquisition software.

Fluorescence Calibration.
State whether fluorescence calibration was implemented, and if so, report the materials and methods used, catalogue numbers, lot numbers, and supplied reference units for the standards.Fluorescence parameters may be reported in standardized units of MESF, ERF, or ABC beads.The type of regression used, and the resulting scatter plot of arbitrary data vs standard data for the reference particles should be supplied.
Fluorescence calibration was performed on PE using FCMPASS software as previously demonstrated [1,2].Details on the calibration reagents and regression can be found in the summary table on the 'Fl Cali' sheet and in herein (FCMPass plots).
State whether and how light scatter calibration was implemented.Light scatter parameters may be reported in standardized units of nm2, along with information required to reproduce the model.
Light scatter calibration was performed using FCMPASS software as previously demonstrated [1,2].Details on the reagents and modelling parameters can be found herein 5.1 EV diameter/surface area/volume approximation.
State whether and how EV diameter, surface area, and/or volume has been calculated using FC measurements.
Diameter approximation was not performed as FCMpass did not have the RI values for virus.Arbitrary units of light scatter were reported as scattering cross section (nm 2 ) 5.2 EV refractive index approximation.State whether the EV refractive index has been approximated and how this was done.
NA-these are not EVs, refractive index approximation was not performed.

EV epitope number approximation.
State whether EV epitope number has been approximated, and if so, how it was approximated.
Epitope approximation was not specifically addressed, however fluorescence data was reported in PE MESF which has a approximated equivalence of 1 epitope to 1 PE MESF.
Complete MIFlowCyt checklist criteria 1 to 4 using the MIFlowCyt guidelines.Template found at www.evflowcytometry.org.
Completion of MIFlowCyt checklist were included.

Calibrated channel detection range
If fluorescence or scatter calibration has been carried out, authors should state whether the upper and lower limits of a calibrated detection channel were calculated in standardized units.This can be done by converting the arbitrary unit scale to a calibrated scaled, as discussed in Section 4.3 and 4.4, and providing the highest unit on this scale and the lowest detectable unit above the unstained population.The lowest unit at which a population is deemed 'positive' can be determined a variety of ways, including reporting the 99th percentile measurement unit of the unstained population for fluorescence.The chosen method for determining at what unit an event was deemed positive should be clearly outlined.
Refer to data presented in FCMPAss Plots in this excel sheet for calibrated detection channel ranges.
State whether EV number/concentration has been reported.If calculated, it is preferable to report EV number/concentration in a standardized manner, stating the number/concentration between a set detection range.
Virus particle concentrations of virus containing supernatants were reported for the gated regions denoted (identified by SSC) in Figure S3.
When applicable, state the method by which the brightness of EVs is reported in standardized units of scatter and/or fluorescence.
Brightness as reported in MESF for fluorescence antibody labeling of cellular proteins was identified in Fig 5, and scattering cross section for light scatter.
7.1.Sharing of data to a public repository.Provide a link to the experimental data in a public data repository.
Data has been shared on flowrepository.org in manuscript data availability section MIFlowCytEv Table

Fig
Fig. S1.Antibody titration of flow virometry antibodies.(pg 1/2) Fig. S3.Comparison of EV versus virus staining (related to Figure 5).Each row shows a set of stains performed on media alone (RPMI), cell culture supernatants from uninfected PBMC (EV control), and HIV BaL virus from PBMC (Virus).Columns show staining with PE-labelled antibodies against CD38, CD44 and Integrin β7.Unstained samples are shown in column one as a control.Particle concentrations of the gated regions are shown in red on each dot plot.