Technical Reference #1789

1789.

Viral mutagenesis as a means for generating novel proteins John N. Davis; Anthony N. van den Pol, Yale University School of Medicine, Journal of Virology, 84(1789), (2010)
Link To Paper

Abstract:
We demonstrate that a mutation-prone virus engineered to express a foreign gene is an expedient means for generating novel mutant nonviral proteins in mammalian cells. Using vesicular stomatitis virus to express a gene coding for a fluorescent DsRed protein a number of green mutant variants including a new variant not previously described were rapidly isolated from infected cells sequenced and cloned. Similar methods may be useful in the development of physiologically sensitive fluorescent reporter proteins and directed evolution or mutagenesis of proteins in general.

Materials & Methods:
To investigate the possibility that viral mutagenesis might lead to functional mutations of foreign non-viral genes we examined over 80 BHK 21 cell cultures infected with VSV-1’DsRed containing 1 hundreds of thousands of red fluorescent plaques and consistently found a small number of green fluorescent plaques (Fig. 1B) 24 hr post inoculation suggesting the presence of mutant DsRed genes. Recombinant VSV-1'DsRed virus (21 24) expressing the DsRed1 gene (Clontech Palo Alto CA) in the first gene position was used (from Drs. J. Rose and I. Simon Yale University). Prior to screening for fluorescent variants plaque purified viral stocks were prepared using single viral plaques to inoculate BHK-21 cells and virus was harvested 24 - 30 hr later. All cells were propagated using MEM growth media consisting of minimum essential medium (Gibco Carlsbad CA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin solution (Gibco) and maintained at 37°C with 5% CO2. Microscopic fluorescent screening and imaging of the DsRed plaques which normally fluoresced red was performed using an Olympus IX71 inverted microscope fitted with DsRed and EGFP filter sets. Quantification of the green variant plaques found in a series of duplicate 35 mm culture wells infected with 3 10 30 and 100 x 103 14 plaque forming units (pfu) per well resulted in the following numbers of green plaques: 3 x 103 pfu = 0 0; 10 x 103 pfu = 0 1; 30 x 103 pfu = 2 3; 100 x 103 16 pfu = 10 12. Thus we find a mean of 1 green variant plaque per 10700 red ones. Individual green fluorescent plaques were identifiable against a background of red fluorescent plaques even at high concentrations of virus up to 100000 pfu/well (Fig. 1C).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
BHK-21 cell cultures