Analysis of herpes simplex virus type I nuclear particles by flow cytometry

Abstract

Flow cytometry has been instrumental to characterize cell populations and examine their inner molecules and processes. In most instances, whole cells are analyzed, and hence, particle size is not an issue. Viruses are 2-3 orders of magnitude smaller than cells so flow cytometry has typically been used to study viral markers within whole infected cells. However, the ability to separate and purify viral particles representing different maturation stages within a viral life cycle would be a useful tool to analyze them in details and characterize the host proteins they associate with. Herpes simplex virus Type 1 is a 250 nm enveloped DNA virus that replicates in the nucleus where it assembles new viral particles called capsids. These capsids eventually travel to the cell surface and are modified along the way, producing several intermediate particles. In the nucleus, three types of stable nonenveloped 125 nm nuclear capsids exist that differ in protein composition and genome content. This includes so-called nuclear C-capsids that are the precursors of mature extracellular virions. We report that we can apply flow cytometry to sort these nuclear C-capsid intermediates by labeling the viral genome with Syto 13, a fluorescent marker that binds to nucleic acids. This is the first time flow cytometry has been used not only to detect but also to purify an intracellular viral maturation intermediate. This opens new research avenues in virology to study capsid assembly, maturation and egress, analyze mutant phenotypes, and define host factors associated with specific viral intermediates. ' 2012 International Society for Advancement of Cytometry

Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles

“…Figure 1A depicts our analysis strategy. As previously reported (24), background particles present in the 0.22-m-filtered phosphate-buffered saline (PBS) shield buffer were detected by light scattering but were otherwise devoid of any intrinsic fluorescence ( Fig. 1Ba and b).…”

“…Since it is not possible to discern 100-m from 200-m particles, whether commercial beads or viruses or the background signal based on light scattering, we circumvented this issue in the past by GFP tagging nuclear capsids or by staining the viral genome with the nucleic acid Syto 13 dye, whose fluorescence is strongly stimulated when bound to nucleic acids. This not only allowed us to tell the capsids apart from the background but also proved to be an efficient tool to efficiently enrich for so-called C nuclear capsids, reaching 90% purity in a single purification step (24).…”

“…The goal of the present study was to quantitatively evaluate the variability of the HSV-1 tegument proteins among viral particles and to evaluate the biological relevance of that particle-to-particle variability. To this end, we resorted to flow cytometry (also referred to as flow virometry), a powerful method we recently employed to characterize and purify HSV-1 nuclear capsids (24). The present study shows that flow cytometry can also be used to study naturally occurring heterologous populations of mature extracellular HSV-1 virions.…”

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

“…Theoretically, the FACS strategy can be used for sorting any other microbes if they have fluorescent markers. Indeed, the FACS strategy has been used successfully in studies of HSV‐1 capsid particles and in synaptosome purification . The successful implementation of the FACS‐based virion purification strategy likely benefited from the large size of PRV (200–250 nm); thus, the effectiveness of this strategy on smaller virions remains to be tested.…”

A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice

“…Virus was collected again by centrifugation and resuspended in PBS, and then analyzed by flow cytometry as described above. Minimum threshold settings on SSC were employed to increase sensitivity for small particles and FSC and SSC parameters were set to log 10 scale as previously described [60]. Deionized water was run for 15 min to equilibrate for low threshold noise.…”