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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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Two-color correlation analysis for EGFP-Vpr and envelope glycoproteins in single HIV-1 virions. (a)–(e) Alexa-594 TPF was plotted as a function of EGFP-Vpr TPF for virions prepared with 0.01, 0.1, 0.2, 2 and 4 µg pEnv, respectively as indicated (the same color code as Figure 5c). (f) The correlation coefficients calculated for each panel from a to e as a function of the pEnv inputs during virion production.
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Figure 6: Two-color correlation analysis for EGFP-Vpr and envelope glycoproteins in single HIV-1 virions. (a)–(e) Alexa-594 TPF was plotted as a function of EGFP-Vpr TPF for virions prepared with 0.01, 0.1, 0.2, 2 and 4 µg pEnv, respectively as indicated (the same color code as Figure 5c). (f) The correlation coefficients calculated for each panel from a to e as a function of the pEnv inputs during virion production.

Mentions: Lastly, the ability to use a single 830 nm laser to excite both EGFP and Alexa-594 simultaneously offers a unique advantage to our technique. Because EGFP and Alexa-594 have minimal emission spectrum overlap (Supplementary Fig. 7), we can resolve both fluorescence for a single HIV-1 particle and thus examine the potential correlation between the two kinds of proteins within a single particle. To this end, we plotted Alexa-594 TPF as a function of EGFP-Vpr TPF for each single HIV-1 virions in Fig. 6a–e for the five batches of viruses that we have prepared with different pEnv inputs. As shown, there was little correlation between Alexa-594 and EGFP-Vpr in their TPF intensities at single-particle level. This conclusion is true regardless of the input quantity of pEnv. The correlation coefficients between Alexa-594 TPF and EGFP-Vpr TPF are 0.1668, 0.2002, 0.1035, 0.3054, 0.1087 for 0.01, 0.1, 0.2, 2 and 4 µg pEnv (Fig. 6f), respectively, suggesting that Vpr and envelope glycoprotein incorporation into individual HIV-1 virions are two independent processes during HIV-1 virion budding.


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)

Two-color correlation analysis for EGFP-Vpr and envelope glycoproteins in single HIV-1 virions. (a)–(e) Alexa-594 TPF was plotted as a function of EGFP-Vpr TPF for virions prepared with 0.01, 0.1, 0.2, 2 and 4 µg pEnv, respectively as indicated (the same color code as Figure 5c). (f) The correlation coefficients calculated for each panel from a to e as a function of the pEnv inputs during virion production.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125448&req=5

Figure 6: Two-color correlation analysis for EGFP-Vpr and envelope glycoproteins in single HIV-1 virions. (a)–(e) Alexa-594 TPF was plotted as a function of EGFP-Vpr TPF for virions prepared with 0.01, 0.1, 0.2, 2 and 4 µg pEnv, respectively as indicated (the same color code as Figure 5c). (f) The correlation coefficients calculated for each panel from a to e as a function of the pEnv inputs during virion production.
Mentions: Lastly, the ability to use a single 830 nm laser to excite both EGFP and Alexa-594 simultaneously offers a unique advantage to our technique. Because EGFP and Alexa-594 have minimal emission spectrum overlap (Supplementary Fig. 7), we can resolve both fluorescence for a single HIV-1 particle and thus examine the potential correlation between the two kinds of proteins within a single particle. To this end, we plotted Alexa-594 TPF as a function of EGFP-Vpr TPF for each single HIV-1 virions in Fig. 6a–e for the five batches of viruses that we have prepared with different pEnv inputs. As shown, there was little correlation between Alexa-594 and EGFP-Vpr in their TPF intensities at single-particle level. This conclusion is true regardless of the input quantity of pEnv. The correlation coefficients between Alexa-594 TPF and EGFP-Vpr TPF are 0.1668, 0.2002, 0.1035, 0.3054, 0.1087 for 0.01, 0.1, 0.2, 2 and 4 µg pEnv (Fig. 6f), respectively, suggesting that Vpr and envelope glycoprotein incorporation into individual HIV-1 virions are two independent processes during HIV-1 virion budding.

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