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Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution.

Pang Y, Song H, Kim JH, Hou X, Cheng W - Nat Nanotechnol (2014)

Bottom Line: Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions.We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level.Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Optical tweezers use the momentum of photons to trap and manipulate microscopic objects, contact-free, in three dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular motors, biopolymers and nanostructures, its application to study viruses has been very limited, largely due to their small size. Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions. We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level. Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

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Optical trapping virometry. (a) Infectivity of HIV-1 virions as a function of pEnv in the presence of 20 µg/ml DEAE-dextran. The error bars are standard deviations from three independent replicates. (b) Alexa-594 TPF as a function of particle diameter. Red circles are for HIV-1 from 2 µg pEnv (N=231) while blue diamonds are for virions without envelope (N=42). (c) TPF intensity histograms for Alexa-594 from single HIV-1 particles, with black, blue, orange, red and olive curves for virions from 0.01 (N=84), 0.1 (N=102), 0.2 (N=100), 2.0 (N=128) and 4.0 µg pEnv (N=76), respectively. (d)–(f) representative Alexa-594 TPF time courses from single virions bound with Alexa594-b12, where individual photobleaching steps in (e) and (f) are indicated with arrows. Insets, cartoons of HIV-1 virions with varied numbers of envelope glycoproteins.
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Figure 5: Optical trapping virometry. (a) Infectivity of HIV-1 virions as a function of pEnv in the presence of 20 µg/ml DEAE-dextran. The error bars are standard deviations from three independent replicates. (b) Alexa-594 TPF as a function of particle diameter. Red circles are for HIV-1 from 2 µg pEnv (N=231) while blue diamonds are for virions without envelope (N=42). (c) TPF intensity histograms for Alexa-594 from single HIV-1 particles, with black, blue, orange, red and olive curves for virions from 0.01 (N=84), 0.1 (N=102), 0.2 (N=100), 2.0 (N=128) and 4.0 µg pEnv (N=76), respectively. (d)–(f) representative Alexa-594 TPF time courses from single virions bound with Alexa594-b12, where individual photobleaching steps in (e) and (f) are indicated with arrows. Insets, cartoons of HIV-1 virions with varied numbers of envelope glycoproteins.

Mentions: We have recently developed single-molecule TPF imaging capability using our optical trap setup34,35. This imaging uses the optical trapping laser for direct and simultaneous TPF excitation of fluorophores such as fluorescent proteins at the laser focus whose fluorescence is detectable at single-molecule level. Because the optical paths for BFP interferometry operate independently from single-molecule TPF detection, we can thus trap a single HIV-1 in culture fluid and simultaneously monitor viral proteins via TPF by using appropriate fluorophore labels. Because our TPF detection has single-molecule sensitivity, this technique should offer many possibilities for quantitation and potential sorting of virions in culture fluid with exquisite sensitivity. To test this idea, we produced series of HIV-1 virions that were tagged with EGFP-Vpr but might carry different number of envelope glycoproteins as we varied the envelope plasmid input (pEnv, 0–4 µg) during virion production30. The infectivity of these virions increased with increasing pEnv (Supplementary Fig. 6) and reached a plateau at 1 µg pEnv in the presence of 20 µg/ml DEAE-dextran (Fig. 5a). To monitor the envelope content of individual virions, we prepared fluorescent-labeled monoclonal antibody b1239 (Alexa594-b12) that specifically recognizes HIV-1 envelope glycoproteins gp12040,41. We chose Alexa-594 for labeling, which can be excited by the 830 nm trapping laser efficiently and yet has a minimal spectral overlap with EGFP emission (Supplementary Fig. 7). This strategy allowed us to measure EGFP-Vpr and gp120 for the same single virion almost simultaneously, both with single-molecule sensitivity34 (Supplementary Fig. 8). Microvesicles that carried gp120 or antibody aggregates were also excluded from HIV-1 virions by using this two-color strategy. We incubated the EGFP-tagged viruses with Alexa594-b12 at an antibody concentration (15 µg/ml) that was sufficient to neutralize >95% HIV infectivity (Supplementary Fig. 9) to ensure the saturation binding of all functional gp120. Control experiments showed that gp120 shedding was negligible at this concentration of b12 (Supplementary Fig. 10). We thus expect the level of Alexa594-b12 fluorescence associated with individual virions to correlate with gp120 content on virion surface. We measured HIV virions prepared with 0, 0.01, 0.1, 0.2, 2 and 4 µg pEnv by trapping them in complete media. A typical result is shown in Fig. 5b using 2 µg pEnv as an example, where the intensity of Alexa-594 TPF for each particle was plotted as a function of particle diameter in red circles (N=231). For 2 µg pEnv, 55% of the particles had diameters that were consistent with single virions and were highlighted in green. The intensity of Alexa-594 TPF associated with these particles displayed a broad distribution, varying from zero to 40,000 a.u.. Control experiments using virions without envelope glycoproteins (pEnv=0) showed no Alexa-594 fluorescence in >95% cases (Fig. 5b blue diamonds), confirming that the different levels of Alexa-594 TPF resulted from specific gp120 binding by Alexa594-b12. Fig. 5c shows a compendium of Alexa-594 TPF histograms from single virions prepared with varied pEnv. Throughout, all the distributions were broad even when the infectivity for the corresponding batch of HIV-1 was at plateau. The single-molecule sensitivity of current setup allowed us to roughly estimate the number of envelope trimers in single virions based on the average TPF intensity of a single Alexa-594 molecule, which yielded 0–18 envelope trimers per virion if assuming each Alexa594-b12 bound two gp120 molecules or 0–9 envelope trimers per virion if each Alexa594-b12 bound only one gp120 (Supplementary Note 3). Indeed, as shown in Fig. 5d, we could clearly distinguish particles that carried gp120 (red curve) from those that didn’t due to single-molecule sensitivity (black curve). 12% of the single particles from 2 µg pEnv did not show any Alexa-594 fluorescence (Fig. 5c), indicating that no gp120 was present on the virion surface. This fraction increased to 26% as we lowered pEnv to 0.01 µg (Fig. 5c). Because envelope glycoproteins are required for virion infectivity (Fig. 5a), these data suggest that defective HIV-1 virions can be produced during virion budding, the fraction of which increases when envelope glycoproteins are supplied under limiting conditions (Supplementary Table 3). Furthermore, there were particles that had gp120 but clearly showed only two or three steps of Alexa-594 photobleaching (Fig. 5e and f). The fraction of these virions was 5% for 2 µg pEnv and increased to 33% for virions prepared with 0.01 µg pEnv. These data indicate that at most a single envelope trimer is present on these virions. The number of envelope trimers per virion was estimated previously using various methods42,43. However, as low as a single trimer on virion surface has not been reliably reported. Our technique allows us to clearly detect the presence of a single trimer on HIV-1 virion surface in culture fluid without ambiguity.


Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution.

Pang Y, Song H, Kim JH, Hou X, Cheng W - Nat Nanotechnol (2014)

Optical trapping virometry. (a) Infectivity of HIV-1 virions as a function of pEnv in the presence of 20 µg/ml DEAE-dextran. The error bars are standard deviations from three independent replicates. (b) Alexa-594 TPF as a function of particle diameter. Red circles are for HIV-1 from 2 µg pEnv (N=231) while blue diamonds are for virions without envelope (N=42). (c) TPF intensity histograms for Alexa-594 from single HIV-1 particles, with black, blue, orange, red and olive curves for virions from 0.01 (N=84), 0.1 (N=102), 0.2 (N=100), 2.0 (N=128) and 4.0 µg pEnv (N=76), respectively. (d)–(f) representative Alexa-594 TPF time courses from single virions bound with Alexa594-b12, where individual photobleaching steps in (e) and (f) are indicated with arrows. Insets, cartoons of HIV-1 virions with varied numbers of envelope glycoproteins.
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Figure 5: Optical trapping virometry. (a) Infectivity of HIV-1 virions as a function of pEnv in the presence of 20 µg/ml DEAE-dextran. The error bars are standard deviations from three independent replicates. (b) Alexa-594 TPF as a function of particle diameter. Red circles are for HIV-1 from 2 µg pEnv (N=231) while blue diamonds are for virions without envelope (N=42). (c) TPF intensity histograms for Alexa-594 from single HIV-1 particles, with black, blue, orange, red and olive curves for virions from 0.01 (N=84), 0.1 (N=102), 0.2 (N=100), 2.0 (N=128) and 4.0 µg pEnv (N=76), respectively. (d)–(f) representative Alexa-594 TPF time courses from single virions bound with Alexa594-b12, where individual photobleaching steps in (e) and (f) are indicated with arrows. Insets, cartoons of HIV-1 virions with varied numbers of envelope glycoproteins.
Mentions: We have recently developed single-molecule TPF imaging capability using our optical trap setup34,35. This imaging uses the optical trapping laser for direct and simultaneous TPF excitation of fluorophores such as fluorescent proteins at the laser focus whose fluorescence is detectable at single-molecule level. Because the optical paths for BFP interferometry operate independently from single-molecule TPF detection, we can thus trap a single HIV-1 in culture fluid and simultaneously monitor viral proteins via TPF by using appropriate fluorophore labels. Because our TPF detection has single-molecule sensitivity, this technique should offer many possibilities for quantitation and potential sorting of virions in culture fluid with exquisite sensitivity. To test this idea, we produced series of HIV-1 virions that were tagged with EGFP-Vpr but might carry different number of envelope glycoproteins as we varied the envelope plasmid input (pEnv, 0–4 µg) during virion production30. The infectivity of these virions increased with increasing pEnv (Supplementary Fig. 6) and reached a plateau at 1 µg pEnv in the presence of 20 µg/ml DEAE-dextran (Fig. 5a). To monitor the envelope content of individual virions, we prepared fluorescent-labeled monoclonal antibody b1239 (Alexa594-b12) that specifically recognizes HIV-1 envelope glycoproteins gp12040,41. We chose Alexa-594 for labeling, which can be excited by the 830 nm trapping laser efficiently and yet has a minimal spectral overlap with EGFP emission (Supplementary Fig. 7). This strategy allowed us to measure EGFP-Vpr and gp120 for the same single virion almost simultaneously, both with single-molecule sensitivity34 (Supplementary Fig. 8). Microvesicles that carried gp120 or antibody aggregates were also excluded from HIV-1 virions by using this two-color strategy. We incubated the EGFP-tagged viruses with Alexa594-b12 at an antibody concentration (15 µg/ml) that was sufficient to neutralize >95% HIV infectivity (Supplementary Fig. 9) to ensure the saturation binding of all functional gp120. Control experiments showed that gp120 shedding was negligible at this concentration of b12 (Supplementary Fig. 10). We thus expect the level of Alexa594-b12 fluorescence associated with individual virions to correlate with gp120 content on virion surface. We measured HIV virions prepared with 0, 0.01, 0.1, 0.2, 2 and 4 µg pEnv by trapping them in complete media. A typical result is shown in Fig. 5b using 2 µg pEnv as an example, where the intensity of Alexa-594 TPF for each particle was plotted as a function of particle diameter in red circles (N=231). For 2 µg pEnv, 55% of the particles had diameters that were consistent with single virions and were highlighted in green. The intensity of Alexa-594 TPF associated with these particles displayed a broad distribution, varying from zero to 40,000 a.u.. Control experiments using virions without envelope glycoproteins (pEnv=0) showed no Alexa-594 fluorescence in >95% cases (Fig. 5b blue diamonds), confirming that the different levels of Alexa-594 TPF resulted from specific gp120 binding by Alexa594-b12. Fig. 5c shows a compendium of Alexa-594 TPF histograms from single virions prepared with varied pEnv. Throughout, all the distributions were broad even when the infectivity for the corresponding batch of HIV-1 was at plateau. The single-molecule sensitivity of current setup allowed us to roughly estimate the number of envelope trimers in single virions based on the average TPF intensity of a single Alexa-594 molecule, which yielded 0–18 envelope trimers per virion if assuming each Alexa594-b12 bound two gp120 molecules or 0–9 envelope trimers per virion if each Alexa594-b12 bound only one gp120 (Supplementary Note 3). Indeed, as shown in Fig. 5d, we could clearly distinguish particles that carried gp120 (red curve) from those that didn’t due to single-molecule sensitivity (black curve). 12% of the single particles from 2 µg pEnv did not show any Alexa-594 fluorescence (Fig. 5c), indicating that no gp120 was present on the virion surface. This fraction increased to 26% as we lowered pEnv to 0.01 µg (Fig. 5c). Because envelope glycoproteins are required for virion infectivity (Fig. 5a), these data suggest that defective HIV-1 virions can be produced during virion budding, the fraction of which increases when envelope glycoproteins are supplied under limiting conditions (Supplementary Table 3). Furthermore, there were particles that had gp120 but clearly showed only two or three steps of Alexa-594 photobleaching (Fig. 5e and f). The fraction of these virions was 5% for 2 µg pEnv and increased to 33% for virions prepared with 0.01 µg pEnv. These data indicate that at most a single envelope trimer is present on these virions. The number of envelope trimers per virion was estimated previously using various methods42,43. However, as low as a single trimer on virion surface has not been reliably reported. Our technique allows us to clearly detect the presence of a single trimer on HIV-1 virion surface in culture fluid without ambiguity.

Bottom Line: Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions.We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level.Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Optical tweezers use the momentum of photons to trap and manipulate microscopic objects, contact-free, in three dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular motors, biopolymers and nanostructures, its application to study viruses has been very limited, largely due to their small size. Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions. We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level. Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

Show MeSH
Related in: MedlinePlus