SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Two-color fluorescence analysis of individual virions determines the distribution of copy number of proteins in herpes simplex virus particles
Richard Clarke 1, Haitao Li 1, Nilah Monnier 2, Dejian Zhou 1, Helena Browne 1 and David Klenerman 3*
1 Cambridge
2 Harvard University
3 Cambridge University
* To whom correspondence should be addressed. E-mail: dk10012{at}cam.ac.uk.
Submitted on February 13, 2007
Revised on March 23, 2007
Accepted on 11 April 2007
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Abstract |
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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 (HSV1). Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently-labelled tegument and envelope proteins relative to a capsid protein by analyzing fluorescence intensity ratios for ensembles of individual dual-labelled 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 gDs 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.
Key Words:
Herpes simplex Virus, Single molecule fluorescence, Two color coincidence detection, glycoprotein
