High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

Bonar, Michał M.; Tilton, John C.

doi:10.1016/j.virol.2017.02.016

Cited by 48 publications

(73 citation statements)

References 55 publications

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“…Nanoscale flow cytometry (NFC), also called flow virometry, is a new and powerful tool in the field of virology that enables the phenotypic analysis of the markers at the surface of individual virions 11 , 25 – 32 . Virus populations can now be profiled and sorted in multi-parameter analyses, much in the same way as cells 25 , 27 , 30 , 32 , 33 . However, NFC analysis with current instrumentation can be challenging due to the fact that these particles are at the limit of detection for flow cytometers.…”

Section: Introductionmentioning

confidence: 99%

“…This can be much lower than the published manufacturer’s specifications, but the onus falls on individual users to achieve this. Despite these caveats, several groups are currently using conventional flow cytometers originally designed for cells to analyze nanoparticles in the 90–150 nm size range 11 , 25 – 34 , 36 – 40 . Common instrument hardware additions for small particle detection include lasers with higher power and the use of photomultiplier tubes (PMTs) instead of photodiodes for forward scatter detection.…”

Section: Introductionmentioning

confidence: 99%

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Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Tang

Renner

Fritzsche

et al. 2017

Sci Rep

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Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cellderived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.Retroviruses, such as the human immunodeficiency virus (HIV), are enveloped RNA viruses that range between 90-150 nm in diameter, depending on the species 1-3 . When nascent virions egress from infected cells, they bear contents of the cytosol (e.g., proteins, mRNAs, miRNAs), as well as a portion of the cell membrane embedded with surface receptors to form the viral envelope [4][5][6] . The phenotypic analysis of host-derived markers on the surface of individual viruses is of considerable interest, as it can provide information on the identity of the specific cell types that are infected in a host. However, a major hurdle in purifying retroviruses for single-particle characterization studies is the removal of EVs that are concomitantly released by the cells 7-11 . EV is a broad term that describes all particles with a membrane bilayer released from cells; these can include exosomes, microvesicles, and apoptotic vesicles [12][13][14][15][16] . Small EVs, that are in the size range of retroviruses constitute a major contaminant of virus preparations as they are biochemically and biophysically similar to retroviruses in terms of their refractive indices, buoyant densities, and surface markers 5,7,[17][18][19] . Additionally, EVs can also package retroviral proteins and RNAs that further complicate discrimination [20][21][22][23][24] .Nanoscale flow cytometry (NFC), also called flow virometry, is a new and powerful tool in the field of virology that enables the phenotypic analysis of the markers at the surface of individual virions 11,[25][26][27][28][29][30][31][32] . Virus populations can now be profiled and sorted in multi-parameter analyses, much in the same way as cells 25,27,30,32,33 . However, NFC analysis with current instrumentation can be challenging due to ...

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Tang

Renner

Fritzsche

et al. 2017

Sci Rep

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show abstract

“…Indeed, most viral suspensions are composed of distinct subpopulations, characterized, for example, by different size characteristics or expression of glycoproteins. Previous studies have shown that these viral subpopulations can demonstrate differences in infectivity (69). Although it is at this stage not possible to determine whether the fraction of mobile HSV-1 virions also presents higher infectivity, the high abundance of GAGs in the extracellular matrix and close to the cell surface could provide the means for HSV-1 diffusion in vivo.…”

mentioning

confidence: 97%

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans

Peerboom

Block

Altgärde

et al. 2017

Biophysical Journal

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Many viruses, including herpes simplex (HSV), are recruited to their host cells via interaction between their envelope glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial attachment is of a multivalent nature, i.e., it requires the establishment of multiple bonds between amino acids of viral glycoproteins and sulfated saccharides on the GAG chain. To gain understanding of how this binding process is modulated, we performed binding kinetics and mobility studies using end-grafted GAG chains that mimic the end attachment of these chains to proteoglycans. Total internal reflection fluorescence microscopy was used to probe binding and release, as well as the diffusion of single HSV-1 particles. To verify the hypothesis that the degree of sulfation, but also the arrangement of sulfate groups along the GAG chain, plays a key role in HSV binding, we tested two native GAGs (chondroitin sulfate and heparan sulfate) and compared our results to chemically sulfated hyaluronan. HSV-1 recognized all sulfated GAGs, but not the nonsulfated hyaluronan, indicating that binding is specific to the presence of sulfate groups. Furthermore we observed that a notable fraction of GAG-bound virions exhibit lateral mobility, although the multivalent binding to the immobilized GAG brushes ensures firm virus attachment to the interface. Diffusion was faster on the two native GAGs, one of which, chondroitin sulfate, was also characterized by the highest association rate per GAG chain. This highlights the complexity of multivalent virus-GAG interactions and suggests that the spatial arrangement of sulfates along native GAG chains may play a role in modulating the characteristics of the HSV-GAG interaction. Altogether, these results, obtained with a minimal and well-controlled model of the cell membrane, provide, to our knowledge, new insights into the dynamics of the HSV-GAG interaction.

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“…Despite these exciting initial reports, the analysis of viruses by flow cytometry, which Grivel and colleagues termed "flow virometry" in 2013 (14), has become more mainstream only recently. Flow virometry has now been used to characterize an expanding array of additional viruses, including lambda phage (15), herpes simplex virus 1 (HSV-1) (16,17), mouse hepatitis virus (MHV) (18), human immunodeficiency virus (HIV) (14,(19)(20)(21), Nipah virus (22), Junin virus (23), vaccinia virus (24), dengue virus (20,25), human cytomegalovirus (HCMV) (26), and giant viruses (13). Flow virometry is becoming a powerful tool to characterize viruses.…”

Section: The Birth Of Flow Virometrymentioning

confidence: 99%

Flow Virometry: a Powerful Tool To Functionally Characterize Viruses

Lippé

2018

J Virol

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For several decades, flow cytometry has been a common approach to analyze cells and sort them to near-purity. It enables one to probe inner cellular molecules, surface receptors, or infected cells. However, the analysis of smaller entities such as viruses and exocytic vesicles has been more difficult but is becoming mainstream. This has in part been due to the development of new instrumentation with resolutions below that of conventional cytometers. It is also attributed to the several means employed to fluorescently label viruses, hence enabling them to stand out from similarly sized particles representing background noise. Thus far, more than a dozen different viruses ranging in size from 40 nm to giant viruses have been probed by this approach, which was recently dubbed "flow virometry." These studies have collectively highlighted the breadth of the applications of this method, which, for example, has elucidated the maturation of dengue virus, served as quality control for vaccinia vaccines, and enabled the sorting of herpes simplex virus discrete viral particles. The present review focuses on the means employed to characterize and sort viruses by this powerful technology and on the emerging uses of flow virometry. It similarly addresses some of its current challenges and limitations.

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High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

Cited by 48 publications

References 55 publications

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans

Flow Virometry: a Powerful Tool To Functionally Characterize Viruses

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