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Reconstitution of potato virus X in vitro

Virology, ISSN: 0042-6822, Vol: 68, Issue: 2, Page: 299-308
1975
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Rapid reconstitution in vitro of infectious potato virus X (PVX) was achieved in mixtures of coat protein subunits and RNA prepared by freezing and thawing purified virus in 2 M LiCl. No reconstitution was obtained with protein subunits prepared with CaCl 2, pyridine, or sodium dodecyl sulphate. The reconstitution reaction occurred at 20°C but not at 4°C, and at low (10 m M ) but not at moderate (100 m M ) salt concentrations; the optimal pH was between 6.0 and 6.2 The structure of reconstituted PVX was shown by optical diffraction analysis of electron micrograph images to be the same as that of native virus. Both reconstituted and native PVX had a basic helical pitch of 3.65 nm that repeated every eight turns along the particle axis, interparticle spacings of 12.6 nm, and no evidence of an axial hole. Reconstitution of PVX in vitro was accompanied by reappearance of the antigenic specificity of intact virus particles. Reconstituted virus particles were heterogeneous in size, and the ribonuclease resistant infectivity was generally less than 10% that of native virus. This may result from trace amounts of ribonuclease contamination in the protein subunit preparations hydrolyzing the RNA before reconstitution of full-length particles occurred.

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