Flow Virometry Research Articles

Implementation and validation of MICaFVi: A highly efficient nanotechnology-based method for coronaviruses detection

Coronaviruses have emerged as a significant public health concern due to the global impact of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which cause COVID-19. The development of sensitive and accurate detection methods is critical for early diagnosis, disease management, and outbreak control. In a previous study, we developed and optimized a nanobased detection methodology called Magnetic Immuno-Capture Followed by Flow Virometry (MICaFVi) using virus-mimicking silica nanoparticles and MERS-CoV/SARS-CoV-2 pseudoviral particles. In the present study, we have extended this methodology to evaluate its specificity and sensitivity for detecting wild-type MERS-CoV and SARS-CoV-2 in human and camel samples. Our results demonstrated that MICaFVi successfully detected MERS-CoV and SARS-CoV-2 viruses with high sensitivity and specificity, although it showed reduced performance for samples with Ct values of 30 or lower compared to qPCR. Despite some limitations in detection speed and sensitivity, MICaFVi represents a significant advancement in diagnostic methodologies by combining nanotechnology with flow cytometry. Additionally, we adapted the MICaFVi methodology to simultaneously detect MERS-CoV and SARS-CoV-2 in a single multiplex assay. The successful implementation of this advanced detection approach has important implications for improving early detection, surveillance, and control of both current and future viral or bacterial pandemics. Our results underscore the potential of MICaFVi as a valuable tool for monitoring the spread of these viruses and highlight its role in advancing diagnostic technologies. This extension of our earlier work offers new insights into the application of nanotechnology and flow cytometry for viral detection.

Applying Flow Virometry to Study the HIV Envelope Glycoprotein and Differences Across HIV Model Systems.

The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.

Analysis of Individual Viral Particles by Flow Virometry.

This review focuses on the emerging field of flow virometry, the study and characterization of individual viral particles using flow cytometry instruments and protocols optimized for the detection of nanoscale events. Flow virometry faces considerable technical challenges including minimal light scattering by small viruses that complicates detection, coincidental detection of multiple small particles due to their high concentrations, and challenges with sample preparation including the inability to easily "wash" samples to remove unbound fluorescent antibodies. We will discuss how the field has overcome these challenges to reveal novel insights into viral biology.

Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells.

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.

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.

Flow Virometry: A Fluorescence-Based Approach to Enumerate Bacteriophages in Liquid Samples.

Correctly designed flow cytometry (virometry) assays allow accurate detection and enumeration of viruses in water. However, rigorous controls and calibrators are needed to obtain quality data. In the absence of proper controls, the use of fluorescent dyes for virus enumeration can produce false positive signals and lead to the wrong estimation of total virus counts by misreporting colloid particles as virions. Here we describe a protocol that addresses the problems that might potentially confound virometry data accuracy.

Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

BackgroundRecombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product.MethodsThe direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads.ResultsUpon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days.ConclusionsAltogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases.Graphical

MICaFVi: A Novel Magnetic Immuno-Capture Flow Virometry Nano-Based Diagnostic Tool for Detection of Coronaviruses.

COVID-19 has resulted in a pandemic that aggravated the world's healthcare systems, economies, and education, and caused millions of global deaths. Until now, there has been no specific, reliable, and effective treatment to combat the virus and its variants. The current standard tedious PCR-based tests have limitations in terms of sensitivity, specificity, turnaround time, and false negative results. Thus, an alternative, rapid, accurate, and sensitive diagnostic tool that can detect viral particles, without the need for amplification or viral replication, is central to infectious disease surveillance. Here, we report MICaFVi (Magnetic Immuno-Capture Flow Virometry), a novel precise nano-biosensor diagnostic assay for coronavirus detection which combines the MNP-based immuno-capture of viruses for enrichment followed by flow-virometry analysis, enabling the sensitive detection of viral particles and pseudoviruses. As proof of concept, virus-mimicking spike-protein-coated silica particles (VM-SPs) were captured using anti-spike-antibody-conjugated MNPs (AS-MNPs) followed by detection using flow cytometry. Our results showed that MICaFVi can successfully detect viral MERS-CoV/SARS-CoV-2-mimicking particles as well as MERS-CoV pseudoviral particles (MERSpp) with high specificity and sensitivity, where a limit of detection (LOD) of 3.9 µg/mL (20 pmol/mL) was achieved. The proposed method has great potential for designing practical, specific, and point-of-care testing for rapid and sensitive diagnoses of coronavirus and other infectious diseases.

Flow virometry for water-quality assessment: protocol optimization for a model virus and automation of data analysis

Flow virometry (FVM) can support advanced water treatment and reuse by delivering near-real-time information about viral water quality. But maximizing the potential of FVM in water treatment and reuse applications requires protocols to facilitate data validation and interlaboratory comparison—as well as approaches to protocol design to extend the suite of viruses that FVM can feasibly and efficiently monitor. We address these needs herein. First, we optimize a sample-preparation protocol for a model virus using a fractional factorial experimental design. The final protocol for FVM-based detection of T4—an environmentally relevant viral surrogate—blends and improves on existing protocols developed using a traditional pipeline-style optimization approach. Second, we test whether density-based clustering can aid and improve analysis of viral surrogates in complex matrices relative to manual gating. We compare manual gating with results obtained through algorithmic clustering: specifically, by leveraging the OPTICS (Ordering Points to Identify Cluster Structure) ordering algorithm. We demonstrate that OPTICS-assisted clustering can work as well or better than manual gating of FVM data, and can identify features in FVM data difficult to detect through manual gating. We demonstrate our combined sample-preparation and automated data-analysis pipeline on wastewater samples augmented with viral surrogates. We recommend use of this protocol to validate instrument performance prior to and alongside application of FVM on environmental samples. Adoption of a consistent, optimized analytical approach that (i) centers on a widely available, easy-to-use viral target, and (ii) includes automated data analysis will bolster confidence in FVM for microbial water-quality monitoring.

Micro-flow imaging and flow virometry as preferred methods to monitor particulate matter for a human cytomegalovirus vaccine candidate.

The presence of particulate matter in vaccine samples is of concern as aggregates have the potential to reduce product efficacy, impact immunogenicity, or block capillaries upon injection. As a result, regulatory agencies routinely request particulate matter tests for particles in the micron size range for release. Particles may come in a variety of forms and can be intrinsic (e.g., from proteinaceous aggregates) or extrinsic (e.g., silicone oil from tubing or contamination from the manufacturing process). They may span the size range of sub-visible to visible particle sizes. In recent years, there has been a rise in requests from regulatory agencies for methods to detect and monitor aggregated particles as well as a desire to evaluate particle size and concentration of sub-micron sized particles. Typically utilized techniques include biophysical analytical methodologies that leverage light scattering or imaging. In this study, Dynamic Light Scattering (DLS), Micro-Flow Imaging (MFI) and Flow Virometry are evaluated as methods to detect and monitor sub-micron to micron sized particles, to support assessments of stability and processability, for drug substance (DS) and drug product (DP) samples of a Human Cytomegalovirus (HCMV) vaccine candidate. For this attenuated live virus vaccine candidate, MFI and flow virometry proved to be the most sensitive methods to monitor particulate matter.

Cytometry and analytical methods in COVID‐19 diagnostics and potential treatment: A special issue on Cytometry Part A

Cytometry and analytical methods in <scp>COVID</scp>‐19 diagnostics and potential treatment: A special issue on Cytometry Part A

Development of process analytical tools for rapid monitoring of live virus vaccines in manufacturing

In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between “good” and “damaged” virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.

Viro-fluidics: Real-time analysis of virus production kinetics at the single-cell level

Real-time visualization and quantification of viruses released by a cell are crucial to further decipher infection processes. Kinetics studies at the single-cell level will circumvent the limitations of bulk assays with asynchronous virus replication. We have implemented a “viro-fluidic” method, which combines microfluidics and virology at single-cell and single-virus resolutions. As an experimental model, we used standard cell lines producing fluorescent HIV-like particles (VLPs). First, to scale the strategy to the single-cell level, we validated a sensitive flow virometry system to detect VLPs in low concentration samples (≥104 VLPs/mL). Then, this system was coupled to a single-cell trapping device to monitor in real-time the VLPs released, one at a time, from single cells under cell culture conditions. Our results revealed an average production rate of 50 VLPs/h/cell similar to the rate estimated for the same cells grown in population. Thus, the virus-producing capacities of the trapped cells were preserved and its real-time monitoring was accurate. Moreover, single-cell analysis revealed a release of VLPs with stochastic bursts with typical time intervals of few minutes, revealing the existence of limiting step(s) in the virus biogenesis process. Our tools can be applied to other pathogens or to extracellular vesicles to elucidate the dissemination mechanisms of these biological nanoparticles.

Functional profiling of Covid 19 vaccine candidate by flow virometry

Vaccine development is a complex process, starting with selection of a promising immunogen in the discovery phase, followed by process development in the preclinical phase, and later by clinical trials in tandem with process improvements and scale up. A large suite of analytical techniques is required to gain understanding of the vaccine candidate so that a relevant immunogen is selected and subsequently manufactured consistently throughout the lifespan of the product. For viral vaccines, successful immunogen production is contingent on its maintained antigenicity and/or infectivity, as well as the ability to characterize these qualities within the context of the process, formulation, and clinical performance. In this report we show the utility of flow virometry during preclinical development of a Covid 19 vaccine candidate based on SARS-CoV-2 spike (S) protein expressed on vesicular stomatitis virus (VSV). Using a panel of monoclonal antibodies, we were able to detect the S protein on the surface of the recombinant VSV virus, monitor the expression levels, detect differences in the antigen based on S protein sequence and after virus inactivation, and monitor S protein stability. Collectively, flow virometry provided important data that helped to guide preclinical development of this vaccine candidate.

Bluetongue Virus Detection Using Microspheres Conjugated with Monoclonal Antibodies against Group-Specific Protein Vp7 by Flow Virometry

Bluetongue Virus Detection Using Microspheres Conjugated with Monoclonal Antibodies against Group-Specific Protein Vp7 by Flow Virometry

Capturing Coronaviruses: Comparison of Recovery Methods for Infectious Coronaviruses From Environmental Surfaces

The emergence of novel, zoonotic betacoronavirus, SARS‐CoV‐2, demands quantification of infectious virus rather than viral RNA to provide more accurate assessment of transmission risk for COVID‐19. To maximize investigator safety, similar in morphology and inactivation profile, human betacoronavirus OC43 (OC43) was used as a surrogate to refine infectious virus recovery methods for SARS‐CoV‐2, a biosafety level (BSL)‐3 pathogen. Using OC43 and SARS‐CoV‐2, we examined the ability of InnovaPrep Mano Surface Samplers (MANOs), made of hydrophobic, open‐cell polymeric foam, to recover virus from large stainless‐steel surfaces hypothesizing that they would outperform hydrophilic cellulose sponges (sponges), used for environmental sampling.In triplicate, 1.0 x 105 TCID50 of OC43 and SARS‐CoV‐2 were applied to the inner surface of a biological safety cabinet (BSC) within the literature or vendor specified optimal surface area, 1267.36 cm2 (MANO) and 100 cm2 (sponges). The areas were sampled with eluant presoaked MANOs and sponges, and samples aliquoted and stored at ‐80ºC until batch titration by indirect immunofluorescence TCID50 assay, using human ileocecal colorectal adenocarcinoma cells (HRT‐18 CCL‐244, ATCC) for OC43 samples and crystal violet detection‐based TCID50 using African green monkey kidney epithelial cells (VeroE6 CRL‐1586, ATCC) for SARS‐CoV‐2. Tested eluants were a beef extract buffer, pH 7 (BEB7), BEB7 with 0.05% Tween‐20 (BEB7/T) and (MANOs only) phosphate buffered saline with 0.05% Tween‐20 (PBS/T), the vendor default. Additionally, an expanded surface area (0.77m2 for MANOs, 300cm2 for sponges) was tested with OC43 as described above, except with 1.0x104 TCID50 inoculum.For OC43, recovery for MANOs with BEB7 was 2.66x105 TCID50 compared to PBS/T’s 4.40x104 TCID50. BEB7 MANOs had a higher average percent recovery (102%) than BEB7 sponges (84%). By one‐way ANOVA (α=0.05), MANO BEB7’s and BEB7/T’s average recovery percentages were significantly greater than PBS/T’s. Samples taken from the larger surface areas for both samplers were below the assay’s limits of detection for BEB7; however, OC43 eluted with BEB7/T from sponges was detectable (3.47x102 TCID50, 32% recovery). For SARS‐CoV‐2, MANO BEB7’s average recovery percentage (27%) was again shown to be significantly greater than PBS/T’s (0.38%). However, decreased recovery of infectious virus was seen across both tools and all eluants, perhaps indicating a difference between SARS‐CoV‐2 and OC43 for BEB7.Use of BEB7 improved OC43 and SARS‐CoV‐2 recovery from optimal surface areas with MANOs. Surprisingly, when the surface area was enlarged, all samples except those from BEB7/T sponges were below the limit of detection. This might be explained by additional eluant loss on the larger surface during mechanical recovery or virus inactivation due to desiccation from BSC airflow or other environmental factors. Similar factors might explain the SARS‐CoV‐2 results. Quantification by RT‐qPCR or flow virometry, and planned experiments in BSL‐3 agricultural space, where the room is primary containment, may provide further insights.

Micrometric DNA/PEI polyplexes correlate with higher transient gene expression yields in HEK 293 cells

DNA delivery with polyethylenimine (PEI) has been widely used in the last three decades for the transfection of mammalian cells. Advances in novel characterization techniques at the nanometric scale offer new opportunities to revisit the physicochemical properties of DNA/PEI polyplexes that lead to efficient transfection. In this work, these properties are tuned by studying the synergies between simple parameters such as NaCl concentration, pH and incubation time in the DNA/PEI polyplex preparation protocol by means of Design of Experiments (DoE). By doing so, a model is obtained where an optimal NaCl concentration of 125 mM and an incubation time of 11 min provided the highest transfection yields. Correlation analyses between the physicochemical properties of DNA/PEI polyplexes and the predicted model responses revealed the existence of an optimal degree of aggregation in the pre-complexing solution to attain the highest transfection efficiencies. The presence of these micrometric DNA/PEI polyplex aggregates was confirmed by several nanoparticle characterization techniques including cryo-TEM, DLS and flow virometry. The findings provide a better understanding of the role of DNA/PEI aggregates in transient gene expression approaches, in particular considering that similar complexation protocols and saline solutions are widely used for the transfection of mammalian cell cultures.

Flow Virometry for Characterizing the Size, Concentration, and Surface Antigens of Viruses.

Application of flow cytometry principles for the analysis of viruses has been referred to as flow virometry (FVM). FVM is a multiparametric, high-throughput, and sensitive technique that allows viral particles to be detected, quantified, and characterized based on the biophysical properties of the virus and the expression of proteins on their surface. More specifically, by calibrating the flow cytometer with reference materials, it is possible to measure the concentration of intact viral particles in a sample, the abundance of a target antigen on the surface of the virus, and the relative diameter of the virus. Here, we describe a comprehensive overview of procedures used to stain, detect, and quantify viral and host-derived proteins located on the surface of retroviruses. These outlined techniques can be applied for the rapid phenotypic characterization of retroviruses, other enveloped viruses, and generally most viruses at the single-particle level through the direct staining of viruses collected from the supernatant of infected cells, without the need for enrichment or purification. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Virus production Basic Protocol 2: Instrument setup, standardization, and quality control for fluorescence quantification Basic Protocol 3: Flow virometry analysis Basic Protocol 4: Viral surface antigen staining and fluorescence quantification Support Protocol: Determination of the optimal antibody concentration for virus staining Basic Protocol 5: Gain configuration optimization.

MCMV Centrifugal Enhancement: A New Spin on an Old Topic.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen infecting a majority of people worldwide, with diseases ranging from mild to life-threatening. Its clinical relevance in immunocompromised people and congenital infections have made treatment and vaccine development a top priority. Because of cytomegaloviruses’ species specificity, murine cytomegalovirus (MCMV) models have historically informed and advanced translational CMV therapies. Using the phenomenon of centrifugal enhancement, we explored differences between MCMVs derived in vitro and in vivo. We found centrifugal enhancement on tissue culture-derived virus (TCV) was ~3× greater compared with salivary gland derived virus (SGV). Using novel “flow virometry”, we found that TCV contained a distinct submicron particle composition compared to SGV. Using an inhibitor of exosome production, we show these submicron particles are not extracellular vesicles that contribute to centrifugal enhancement. We examined how these differences in submicron particles potentially contribute to differing centrifugal enhancement phenotypes, as well as broader in vivo vs. in vitro MCMV differences.

Influence of GlycoGag on the Incorporation of Host Membrane Proteins Into the Envelope of the Moloney Murine Leukemia Virus

Analysis of viral particle heterogeneity produced from infected cells has been limited by the inefficiency of traditional analytical methods to characterize large populations of viruses at an individual particle level. Flow virometry (FVM) is an emerging technique based on flow cytometry principles that enables a high throughput, multiparametric, and phenotypic characterization of viruses at a single particle resolution. Here, we performed FVM to analyze surface markers found on Murine Leukemia Virus (MLV) and glycosylated Gag-deficient (glycoGag) MLV. The glycoGag viral accessory protein has several roles in the MLV viral infection cycle including directing retroviral assembly and particle release at lipid rafts. Based on previous studies, we hypothesize that glycoGag modulates host protein incorporation into the viral envelope during viral assembly and budding. Here, by using FVM, we reveal that glycoGag is associated with an increased incorporation of the host-derived tetraspanins CD81 and CD63 along with the lipid raft marker and immune antigen Thy1.2 during the assembly and release of viral particles from infected NIH 3T3, EL4, and primary CD4+ T cells. Moreover, we show differences in the uptake of host proteins by viruses that are released from the two cell lines and primary T lymphocytes. Additionally, at the individual viral particle level, we observed a degree of expression heterogeneity of host-derived antigens within the viral population. Finally, certain cellular antigens can show either enrichment or exclusion from the viral envelope depending on whether glycoGag is expressed by the virus. This suggests that glycoGag is involved in a mechanism of selective host protein incorporation into the viral envelope.