title: Two-color fluorescence analysis of individual virions determines the distribution of the copy number of proteins in herpes simplex virus particles.
abstract: We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to a capsid protein by analyzing fluorescence intensity ratios for ensembles of individual dual-labeled virions and fitting the resulting histogram of ratios. Using EYFP-tagged capsid protein VP26 as a reference for fluorescence intensity, we are able to calculate the mean and also, for the first time to our knowledge, the variation in numbers of gD, VP16, and VP22 tegument. The measurement of the number of glycoprotein D molecules was in good agreement with independent measurements of average numbers of these glycoproteins in bulk virus preparations, validating the method. The accuracy, straightforward data processing, and high throughput of this technique make it widely applicable to the analysis of the molecular composition of large complexes in general, and it is particularly suited to providing insights into virus structure, assembly, and infectivity.
authors.affiliation: Department of Chemistry and Division of Virology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Clarke
authors.forename: Nilah
authors.initials: N
authors.lastname: Monnier
authors.forename: Haitao
authors.initials: H
authors.lastname: Li
authors.forename: Dejian
authors.initials: D
authors.lastname: Zhou
authors.forename: Helena
authors.initials: H
authors.lastname: Browne
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
mesh_terms: D000906:Antibodies / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives; D005453:Fluorescence; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014759:Viral Envelope Proteins / Q000276:immunology / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
chemical_list: D000906:Antibodies; D036022:Capsid Proteins; D005456:Fluorescent Dyes; D017405:Herpes Simplex Virus Protein Vmw65; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C077409:capsid protein VP26, herpes simplex virus type 1; C485184:enhanced green fluorescent protein; C039447:glycoprotein D, Human herpesvirus 1; C073413:herpes simplex virus type 1 protein VP22; D049452:Green Fluorescent Proteins; C067297:8-aminohexylamino cAMP; D000242:Cyclic AMP