Overexpression of PSGL-1 in HEK293T cells markedly reduces infectivity of progeny virions, while viruses produced by natural infection of T cells and PBMC retain infectivity

In our recent work we phenotyped pseudoviruses engineered to display the virion-incorporated cellular proteins integrin α4β7, CD14 and PSGL-1 (CD162). This work led us to the striking observation that PSGL-1 was detected at markedly higher levels on virion surfaces compared to other host proteins, despite all pseudoviruses being produced with equal amounts of pDNA for host protein expression [28]. Since we observed PSGL-1 to be incorporated to a greater extent than other host proteins on the surface of pseudoviruses, we wanted to determine how phenotypically similar viruses produced through transfection were to viruses produced in more physiologically relevant model systems, such as infection of T cell lines and primary cells.

To this end, we chose to generate viruses with three different cellular models, either through infection or transfection methods (Fig. 1a). We first produced HIV-1 pseudovirus (PV) stocks via co-transfection of HEK293T cells with plasmids expressing an HIV-1 backbone and envelope (SG3ΔEnv backbone + BaL.01 envelope), together with a PSGL-1 expression plasmid (designated as PV + PSGL-1; Fig. 1b). We also generated a matched viral stock without any PSGL-1 (designated as PV; Fig. 1b). For comparison to these viruses produced through transfection, we infected CD4 + T cell lines (H9 and Jurkat) and PBMC from two different donors using IIIB virus stocks and used the progeny viruses for comparative analyses. Before performing downstream analyses of the viruses, we first assessed the levels of PSGL-1 on all of our virus producer cells through cell surface staining with an anti-PSGL-1 antibody and flow cytometry (Fig. 1b). As expected, high levels of PSGL-1 were present on HEK293T cells transfected to express the protein, whereas cells transfected without PSGL-1 pDNA had levels of PSGL-1 that were similar to the isotype staining control. Cells from the H9 and Jurkat T cell lines as well as those from both PBMC donors also displayed appreciable levels of PSGL-1, suggesting that the PSGL-1 was readily available on cells to be incorporated into budding progeny virions. Next we assessed the infectivity of the viruses generated from these different virus production models with the commonly used TZM-bl reporter cell line, using luminescence as a readout for viral infection [12, 29,30,31]. All virus infections were performed with equal viral inputs (normalized p24 input) to allow for comparisons of infectivity across the virus models. As expected, all of the viruses prepared via infection of T cell lines and PBMC were infectious, with virus produced in Jurkat cells being most infectious. Notably, the pseudovirus produced via transfection without PSGL-1 displayed similar levels of infectivity to the Jurkat virus, while the pseudovirus that was produced in cells which were co-transfected to overexpress PSGL-1 displayed complete abrogation of infection (Fig. 1c).

Fig. 1

Comparison of PSGL-1 cell surface expression and virus infectivity across model systems. A Schematic depicting the three model systems (HEK293T transfection, T cell line infection, PBMC infection) used to produce virus with various amounts of PSGL-1 in the manuscript. B Cell surface expression of PSGL-1 on HEK293T cells 48 h after co-transfection with HIV-1 pseudovirus constructs alone (PV; grey histogram), or with pseudovirus constructs and 250 ng of a vector encoding PSGL-1 (PV + PSGL-1, blue histogram). Endogenous levels of PSGL-1 detected by cell surface staining and flow cytometry analysis on the T cell lines, H9 (green) and Jurkat (purple), or activated peripheral blood mononuclear cells (PBMC) from two different donors (red and pink histograms) used for HIV-1 propgation. Isotype staining is shown with unshaded histograms. C Viruses produced in the transfected HEK293T cells or HIV IIIB-infected T cell lines and PBMC from B were normalized for equal viral p24 input in TZM-bl cell cultures. After 48 h the level of infectivity was measured using luminescence readout (relative light units; RLU). Results displayed are the merged mean ± SEM of three independent experiments with each condition tested in duplicate wells. P values were determined using an unpaired t test (****P < 0.0001)

Since many variables could be responsible for the differences in infectivity seen between viruses produced in different producer cells, we designed a more controlled experiment to confirm if differences in PSGL-1 surface expression were responsible for differences in viral infectivity. To this end, we employed a transfection model to simulate a range of different PSGL-1 expression levels on the cell surface of a single virus producer cell type, namely HEK293T cells. As a control, we first produced pseudoviruses completely devoid of PSGL-1 (PSGL-1Neg) through co-transfection of only the HIV plasmids, as previously described. For the PSGL-1-containing viruses, in addition to HIV expression plasmids, we also co-transfected different amounts of PSGL-1 pDNA (Additional file 1: Table S1) to yield virus progeny with low, medium and high levels of PSGL-1 in the HIV-1 envelope (designated PSGL-1Low, PSGL-1Med, and PSGL-1High, respectively). Before assessing virus infectivity, we first assesed the cell surface expression of PSGL-1 on the virus producer cells (HEK293T) using flow cytometry (Fig. 2a) As expected the PSGL-1Neg cells did not contain show any PSGL1-1 staining, while the cells transfected to display low, medium and high amounts of PSGL-1 displayed increasing fluorescence intensity as the amount of PSGL-1 pDNA increased. Of particular note, the ‘high’ designation in PSGL-1High virions simply represents the highest amount of pDNA used in our model system, but notably it is an amount of pDNA that is commonly used in our group and others in the field who are studying transient protein expression (250 ng pDNA per well). We also generated viruses with ‘medium’ and ‘low’ levels of PSGL-1 expression, via step-wise reductions in PSGL-1 pDNA (25 and 2.5 ng, respectively).

Fig. 2

Titration of PSGL-1 expression on virus producing cells and the effect on virus infectivity. A Cell surface expression of PSGL-1 on HEK293T cells as detected by flow cytometry 48 h after co-transfection with HIV-1 pseudovirus constructs and increasing amounts of PSGL-1 pDNA (as outlined in Additional file 1: Table S1). Isotype staining is shown with empty histograms, and PSGL-1 staining is shown with filled, coloured histograms (blue or gray). B Semi-quantitative comparisons of virion-incorporated PSGL-1 on pseudovirus stocks via immunoblot analysis. The viral capsid protein p24 was used as loading control to ensure equal loading of total virus lysates across all lanes. This immunoblot is representative of three blots performed showing similar results. C Densitometric quantitation of immunoblot data from B. D Virus infection was tested via normalized viral inputs (displayed as 1:1 in graph), followed by three-fold serial dilutions of viruses. All diluted virus stocks were incubated with TZM-bl reporter cells for 48 h before infectivity was measured using luminescence readout (relative light units; RLU). E Infectivity from the RLU reading with the most concentrated amount of virus (1:1) is shown. For C and E the results of unpaired t tests with Bonferroni correction are shown (*P < 0.05; **P < 0.01). Results show the mean ± SEM of three independent experiments

To verify that our virus production system could generate virions with differential amounts of PSGL-1, we assessed the levels of PSGL-1 associated with total virus lysates using Western blot (Fig. 2b). As expected, increasing amounts of PSGL-1 were observed in the virion lysates that correlated directly with increasing amounts of PSGL-1 pDNA used in the virus preparations (i.e., transfections). This was also apparent through the densitometric quantification of the blots (Fig. 2c).

Next, to compare infectivity of the different virus stocks, TZM-bl cells were incubated with serial dilutions of normalized viral input (Fig. 2d). As expected, the infectivity of all viruses generated through transfection was greatly diminished when PSGL-1 was present at high levels within virions (Fig. 2d; PSGL-1High). A dose-dependent potency of PSGL-1 as an antiviral factor was evident in the pseudoviruses, which showed a clear stepwise decline in infectivity as increasing amounts of PSGL-1 pDNA were used to produce virions (Fig. 2d). Remarkably, even the PSGL-1Low pseudovirus, which was created with just 2.5 ng of PSGL-1 pDNA, displayed reduced infectivity compared to PSGL-1Neg pseudovirus (Fig. 2d), suggesting a strong mechanism of selectivity for incorporating this protein into virion envelopes. Statistical analyses of the viral infectivity at the most concentrated virus dilution (1:1) showed significant differences between the control virus without PSGL-1 (PSGL-1Neg) and the PSGL-1Med and PSGL-1High viruses (Fig. 2e).

Since immunoblotting only assesses total virion lysates and cannot distinguish proteins that are displayed on the virion surface from the virion interior, our next steps were to employ two other complementary techniques, virion capture and flow virometry, both of which can specifically assay proteins on the virion surface.

Virions display differential amounts of PSGL-1 and gp120 incorporation dependent on their method of production

To semi-quantitatively assess the amount of PSGL-1 in the envelopes of our different virus preparations, we used a previously described immunomagnetic virion capture assay [24, 28, 32, 33]. This technique was chosen for its ability to selectively assay proteins on the viral surface [14, 34,35,36]. To compare the relative amounts of PSGL-1 and gp120 present on our range of viruses, we performed antibody-mediated virion capture on viral stocks that were normalized for viral input (equal virus p24) across all viruses tested. We compared virus capture with an anti-PSGL-1 monoclonal antibody (mAb) versus an anti-gp120 mAb (Fig. 3). Through the use of normalized virus inputs across all capture reactions, direct comparisons between the amount of antibody-mediated capture can reflect the relative levels of virion incorporated PSGL-1 and gp120. As expected, the anti-PSGL-1 antibody captured all of the pseudovirus (PV) preparations engineered to contain PSGL-1 (PSGL-1Low, PSGL-1Med, PSGL-1High), while no PSGL-1 capture was present with control viruses (PSGL-1Neg) that were devoid of PSGL-1 (Fig. 3a). Stepwise differences in levels of PSGL-1 capture were seen in the PSGL-1-positive pseudoviruses based on their engineered designations (low, medium, high; Fig. 3a). As expected, all of the viruses designed to express PSGL-1 were considered statistically different from the PSGL-1Neg virus. These results were in accordance with what was observed with the immunoblot for PSGL-1 incorporation into pseudoviruses (Fig. 2b). Importantly, virion capture with the anti-gp120 mAb was markedly lower than capture with the anti-PSGL-1 mAb for all pseudovirus phenotypes tested and was particularly low for the PSGL-1Med and PSGL-1High viruses. Indeed, gp120 is normally expected to be present at low levels on circulating virions (8–14 spikes per virion) [37] and PSGL-1 is also known to inhibit Env incorporation into virions [11, 15], so it is unsurprising that gp120 is less abundant on viruses produced from cells transfected to overexpress PSGL-1. In line with this, anti-PSGL-1 capture on viruses produced in T cell lines and PBMC demonstrated that PSGL-1 was present on all of the isolates tested, with a moderate range of variation (Fig. 3b and c). Interestingly, levels of PSGL-1-mediated virion capture were much lower in the more physiologically relevant viruses (from T cell line and PBMC) than pseudoviruses (up to 70-fold less).

Fig. 3

Semi-quantitative comparisons of virion-incorporated PSGL-1 and gp120 on virus stocks via virion capture assay. A Virion capture assays were performed with immunomagnetic beads armed with anti-PSGL-1 or anti-gp120 with normalized inputs (5.25 ng of p24 per capture condition) of pseudovirus (HEK293T), B T cell line viruses, and C PBMC viruses (NL4-3, IIIB, BaL generated in donors 1, 2 and 3 respectively). Bead-associated virus was lysed and HIV-1 p24 Gag was quantified using p24 AlphaLISA as an indicator of the amount of virus capture. Results show the mean ± SEM of three independent experiments in which each condition was tested in duplicate. The results of unpaired t tests with Bonferroni correction are shown (**P < 0.01) for A. Levels of background capture as detected using an isotype control antibody were subtracted from the displayed values

While our virion capture assays confirmed that there were differences between the levels of PSGL-1 on viruses produced in different cell types, the technique is only semi-quantitative and reports on the average phenotype of the whole virus sample. Furthermore, since both virion capture and immunoblot are ‘bulk techniques’, they lack the resolution to interrogate a heterogenous sample containing phenotypically distinct virions with individual variation in the amounts of host and viral proteins. To gain more quantitative data on the abundance of PSGL-1 on individual virus particles, we decided to stain virus particles and analyze by flow virometry, for its advantages in providing high throughput, single virion analyses in a calibrated and quantitative readout [38,39,40,41].

Flow virometry analyses indicate that T cell and PBMC viruses incorporate lower amounts of PSGL-1 than viruses produced in cells transfected to express PSGL-1

We have previously provided detailed methodology showing how HIV-1 can be detected by light scatter on sufficiently sensitive cytometers [28, 42], and that virion-incorporated host proteins (including PSGL-1) can be quantified in the HIV-1 envelope of pseudoviruses using flow virometry [28]. More specifically, by using fluorescent and light scatter calibration reference materials along with calibration software, arbitrary light intensities from stained virus samples can be calibrated and expressed in standard units allowing for quantification and comparison of proteins on virions. These standardization methods allow us to draw close estimates of the total number of antibodies bound to each virion, which can be used as a proxy for total number of proteins on individual viral particles [28]. Of note, and in line with the scope of this study, we focused our efforts on PSGL-1 staining and quantitation, and not HIV-1 Env, since the detection of gp120 is currently below our assay threshold with conventional fluorescent labelling.

To begin, we performed labelling of pseudoviruses that we knew contained high levels of PSGL-1 in the envelope with an R-phycoerythrin (PE)-labelled anti-PSGL-1 mAb (same antibody clone used in virion capture assays). We observed stepwise increasing amounts of median PSGL-1 PE labelling (as reported in calibrated units of molecules of equivalent soluble fluorophore; MESF) on the pseudoviruses generated through transfection (PV; Fig. 4a). The grand median MESF values generated from three biological replicates are shown in Additional file 1: Fig. S1. To determine quantitative MESF staining values, viruses were first gated by side scatter (gate shown in Fig. 4) and then a secondary gate spanning the same SSC profile (not shown) was placed on the population of viruses that were above the threshold of detection (i.e., above background fluorescence; ~ 10 MESF) to generate MESF statistics. This ensured that only viruses that could be visibly distinguished from background signals were contributing to our MESF counts. The pseudoviruses showed a dynamic range of staining, with MESF values as 33 ± 1.2 SD, 93 ± 2.4 SD and 145 ± 2.3 SD (Fig. 4a, top row), for the PSGL-1-Low, -Med and -High viruses, respectively. As expected, control viruses that were devoid of PSGL-1 (PSGL-1Neg) did not exhibit positive staining above our limit of fluorescence detection and fell within the instrument background fluorescence. Additionally, a cell culture medium control sample (Media) stained with the same antibody also did not display any appreciable fluorescence.

Fig. 4

Detecting PSGL-1-incorporation on the surface of viruses using flow virometry. A Staining of pseudoviruses produced through transfection of HEK293T cells with different amounts of PSGL-1 DNA (0 ng, 2.5 ng, 25 ng, 250 ng for negative, low, medium, and high phenotypes, respectively) with a PE-conjugated anti-PSGL-1 antibody. The horizontal dotted line on the virus dot plots denotes background fluorescence and the limit of instrument detection (~ 10 MESF). B PSGL-1 staining of IIIB viruses produced in the H9, Jurkat, A3R5.7 and PM1 T cells. C PSGL-1 staining of HIV IIIB and NL4-3 viruses propropagated in four independent primary cell (PBMC) donors (D1–D4). Data shown are representative of three replicates for each virus stock

Having validated that our flow virometry protocol was effective and sensitive enough to detect differences in surface levels of virion-incorporated PSGL-1 in pseudoviruses, we next acquired our T cell line and PBMC viruses (Fig. 4b and c). Since we anticipated that staining endogenous levels of PSGL-1 on viruses produced through infection would be more challenging to detect than staining on our pseudovirus model, we decided to test additional viruses for both model systems (T cell line and PBMC). To this end we included additional IIIB viruses propagated in the A3R5.7 and PM1 T cell lines, since they produced high titre virus that was readily detectable as monodisperse viral populations on our cytometer by light scatter. For viruses produced in primary cells, we increased our sample size to four donors and tested two NL4-3 isolates and two IIIB that were passaged in cells from independent donors. We observed that viruses produced in T cell lines produced more homogenous virus populations than those seen in viruses made in PBMC, as expected given the nature of the culture conditions. T cell line viruses were more monodisperse than viruses propagated in PBMC and produced ‘cleaner’ dot plots with less background attributable to non-virus events. The heterogeneity in PBMC virus scatterplots compared to those of T cell line viruses is likely attributable to differences in extracellular vesicles (EV) produced by cell line versus PBMC cultures (Fig. 4b vs c). Visible PSGL-1 staining was present on all of the viruses produced in T cell lines (Fig. 4b), with the H9 IIIB virus displaying higher levels of staining than the IIIB isolate produced in Jurkat cells, which mirrors the results seen in our virion capture assay (Fig. 3b).

While the majority of the viral isolates produced via infection of T cells and/or PBMC displayed low levels of staining that were visible by eye (Fig. 4b and c), the MESF values were far more modest than those observed on our pseudoviruses, with most reported around the range of 15 MESF (Additional file 1: Figure S1). Notably, the pseudoviruses were produced with stepwise increases in pDNA, with 2.5, 25 and 250 ng of pDNA per well for the Low, Med, and High PSGL-1 viruses. To determine if we could generate viruses through HEK293T transfection that were more similar to the viruses produced through infection, we generated additional virus stocks that were produced through co-transfection with 10- and 100-fold less PSGL-1 pDNA than what was used for the PSGL-1Low virus (0.25 and 0.025 ng; Additional file 1: Figure S2). As expected, these additional viruses displayed much lower levels of PSGL-1 staining, in a range that was more similar to what was seen on viruses from primary PBMC in Fig. 4c.

Since we were able to readily detect PSGL-1 on all three of the PBMC viruses tested using virion capture assays, but lower levels of detection were observed when staining in flow virometry with the same antibody clone (Fig. 4c), we anticipated that PSGL-1 on some primary virions were at levels that were simply too low to detect using our current flow virometry protocols. Indeed, since our capture assays showed that the amount of PSGL-1 on virions produced in T cell lines and PBMC was similar in magnitude to that of gp120 on the viruses tested (i.e., < three-fold difference; Fig. 3c and d), and since we know that anti-gp120 labelling is currently at the cusp of our detection sensitivity for our flow cytometer (roughly ~ 10 PE MESF which is approximately equivalent to ~ 10 molecules per virus) [28], these data are in line with the limit of detection that we would expect using the flow virometry method.

Importantly, our data suggest that contaminating extracellular vesicles that may be present in flow virometry assays do not appear to hinder staining efficiency and/or the reproducibility of our results generated from complementary techniques. However, since vesicles can fall within the range of our virus scatter gate, they may contribute to quantitative assessments ascribed to virus populations. To assess the contribution of extracellular vesicles to our virus populations, we generated matched (mock infected) cell cultures in HEK293T cells, T cell lines and PBMC for comparison to our viruses produced in the same cell types. To this end we transfected HEK293T cells with the same amounts of PSGL-1 pDNA used to generate viruses but substituted viral plasmids for an empty vector. To produce matched controls for viruses produced through infection, T cell lines and primary cells were mock-infected for the same duration of time used in our regular infection protocols (7–10 days). The cell culture supernatants from all three cell types were then stained and acquired as described for the virus preparations (Additional file 1: Figure S3). Unsurprisingly, all of the cell types had extracellular vesicles present from mock infection and/or mock transfection. Cells that were transfected with the highest levels of PSGL-1 pDNA generated the highest levels of PSGL-1-containing EVs. While these vesicles fell within the broad gate that we set for analysis of virus particles, it is important to note that the virus populations displayed different scattering profiles from vesicles, as detected through comparisons of their side scattering profiles with histogram overlays (Additional file 1: Fig. S3). As expected, mock-infected supernatants from T cell lines produced ‘cleaner’ dot plots with lower levels of vesicle staining than what was seen in primary cell cultures, although it should be acknowledged that by simply infecting cells, we expect that the profile of vesicle production would be altered from that of uninfected cells. In summation, while at this time we cannot effectively remove the contributions of EV staining from our virus staining, it is clearly evident that the EV and virus populations are distinct and distinguishable in scatter and staining profiles.

PSGL-1 is incorporated by a broad range of HIV-1 and SIV isolates and is present on virions in plasma from HIV-infected patients

After demonstrating that there were large differences in the levels of PSGL-1 present on virions produced in different cell types, we next wanted to determine how abundant PSGL-1 was on a broader range of HIV-1 isolates grown in primary PBMC and in clinical samples. Our earlier virus capture experiment had given an indication that levels of incorporation could vary ~ four-fold based on the PBMC donor and viral isolate tested (Fig. 3c), but we sought out to perform a more thorough test. To determine the breadth of PSGL-1 virion-incorporation, we used immunomagnetic virion capture as above to compare PSGL-1 and gp120 incorporation among a panel of lab-adapted and clinical HIV-1 isolates representing different co-receptor usage phenotypes and clades (Table 1), similar to our previous reports for virion-incorporated integrin α4β7 [24]. The different viral isolates were tested undiluted in virion capture assays to determine the potential for PSGL-1 detection at a broad range of viral titres. The PSGL-1 antibody successfully captured all strains of HIV-1 tested, and in most cases at higher levels than anti-gp120 capture. Furthermore, the levels of PSGL-1 capture were independent of HIV-1 clade or co-receptor usage. We compared the relative amount of virion incorporated PSGL-1 to gp120 among the different viral isolates, by calculating the ratio of virion capture with anti-PSGL-1 mAb to anti-gp120 mAb (Table 1, ratio). Across most isolates tested, we observed an appreciable excess of virion-incorporated PSGL-1 relative to gp120, with a ratio average of 4.0 among the 12 replication competent viruses tested. To permit comparisons of these data to the levels of PSGL-1 and gp120 on pseudoviruses, we added the values from our pseudovirus capture to the table (Table 1; HIV-1 PV). In contrast to the PBMC viruses, the ratios on pseudoviruses engineered to be PSGL-1High and PSGL-1Med were 302 and 172, respectively, highlighting several orders of magnitude difference in these model systems for PSGL-1 incorporation. Notably, the PSGL-1Low PV had a ratio that was more similar to that seen with PBMC viruses, demonstrating that physiological levels of PSGL-1 incorporation can be displayed on pseudoviruses produced through transfection when using very low levels of pDNA (< 2.5 ng).

Table 1 Virion incorporation of PSGL-1 and gp120 in a panel of clinical and laboratory virus isolates

Notably, we also detected the presence of PSGL-1 at high levels in two SIV isolates (Table 1), providing further support to the notion that PSGL-1 may be a broad-spectrum host restriction factor [11, 13, 14]. Despite the ability for PSGL-1 to sequester gp41 and to disrupt envelope incorporation into virions [11, 15], all of the HIV-1 and SIV isolates tested displayed detectable levels of gp120 capture, above background as determined with capture using an isotype-matched antibody as a negative control. This may suggest that the viral accessory proteins in primary isolates may be sufficient to counteract the inhibitory effects generated by virion-incorporated PSGL-1 and permit higher levels of gp120 incorporation. It is worthwhile mentioning that while the ratio of PGSL-1:gp120 is quite different in our transfection-based virus models versus the infection-based virus models, the total efficiency of PSGL-1 capture was more similar between some of the primary isolates and the PSGL-1Med virus. Similarly, while our pseudoviruses differ greatly from primary isolates in gp120 capture, the level of gp120 capture could vary through the use of a different monoclonal Ab.

After determining that virion incorporated PSGL-1 was present on all of the virus stocks produced in vitro, we sought to determine whether PSGL-1 could be identified on virions that circulate in viremic, HIV-infected individuals, as to our knowledge, this has yet to be described in the literature. To this end, we assayed virions in plasma samples from 12 patients at variable stages of HIV infection (acute/early to chronic) using immunomagnetic virus capture (Fig. 5). For this test, we used undiluted virus samples to allow us to observe the range of capture efficiency based on variable viremia levels (Additional file 1: Table S2). To increase the sensitivity and to enhance the success of virion capture from patient plasma samples which contain many inherent factors that can hinder this assay (e.g., plasma proteins, extracellular vesicles, etc.), we employed a slightly modified capture assay using biotin-conjugated antibodies and compatible microbeads, which have shown enhanced levels of virion capture in our hands over the conventional protein-G Dynabead captures used earlier in this study. Among the 12 patient plasmas tested, all patients harbored virus with incorporated PSGL-1 with variable efficiency of incorporation (Fig. 5). As an additional control, we chose to assess CD44 incorporation in parallel in the same plasma samples, as CD44 was previously described to be incorporated with high efficiency into HIV-1 virions [43,44,45]. CD44 was also incorporated into virions from all patient plasmas tested, and we observed a wide range of virus input recovered by capture with anti-CD44 mAb. Antibodies against CD44 and PSGL-1 were both found to capture virions within patient plasma at levels that were significantly higher than levels of capture with an isotype control antibody. However, no significant differences were seen between the levels of virus capture when targeting either of these two proteins (PSGL-1 or CD44) among the 12 samples tested. We observed a wide range of viral phenotypes and have included the percentage of input virus captured for each respective antibody in Additional file 1: Table S2 for more detailed characterization. Despite the patient variability, PSGL-1 was present on all clinical samples tested, including those from both the acute/early and chronic stages of infection. However, no statistical differences were found between the amount of captured virus between acute and chronic stages of infection within the small size of our study.

Fig. 5

Virions circulating in vivo in HIV-infected patients contain PSGL-1 and CD44. A Plasma samples from viremic HIV-infected patients ranging from acute/early to chronic stages of HIV-1 infection were tested in virion capture assays using an isotype control antibody (IgG control), anti-PSGL-1 or anti-CD44. Captured virus was lysed, followed by RNA extraction and quantitative real-time PCR for the detection of HIV-1 genome equivalents (in RNA copies/mL). The sample median for each antibody capture condition is displayed along with results of a Mann–Whitney test (***P < 0.001). Bonferroni correction was used for adjustment of statistical significance. Each unique symbol represents a different patient, with open circles denoting patients in the acute/early stage infection and filled circles as patients in the chronic stage of infection

Viruses with incorporated PSGL-1 can be captured by P-selectin and subsequently transferred to target cells for HIV-1 infection

After quantifying virion-incorporated PSGL-1 on a variety of virus types and confirming that PSGL-1 was present on viruses from clinical samples, we next decided to assess whether virion-incorporated PSGL-1 was able to maintain its inherent biological functions and bind its cognate receptors, the selectin family members. We speculated that this was highly plausible, since several other virion-incorporated proteins have been reported to maintain their physiological functions [16,