PURIFICATION AND MOLECULAR ANATOMY OF THE VARICELLA-ZOSTER VIRION

Abstract

Varicella-zoster virus (VZV) infected human melanoma cell cultures were harvested and sonically disrupted when cytopathic effect was advanced. Infectious cell-free virus in the sonicates, as well as that in the culture medium, was further concentrated by precipitation with 8% (wt/vol) polyethylene glycol in the presence of high salinity (0.5 M). The virus-enriched pellet was layered onto 15-45% linear metrizamide gradients and sedimented for 18 h at 70,000 g. Of the 3 visible bands (designated upper, middle and lower), the middle band at a buoyant density of 1.156-7 g/cm3 was enriched for enveloped virions. EM enumeration of particles demonstrated a total of 10.04 log10 enveloped particles and 8.26 log10 unenveloped particles from middle bands representing the yield from a 150 cm2 VZV-infected monolayer. Fractionation of radiolabeled virion preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 30 polypeptides between 30 and 200 kilodaltons (K) with a total MW of 2,240,000. Prominent structural polypeptides included the major capsid protein (155K) and 3 glycoproteins, 62K, 98K and 1118K. Certain polypeptides better labeled by [14C]amino acids than by [35S]methionine included a higher MW polypeptide (174K) and 45K protein comigrating with actin. Immune precipitation of a Nonidet-extracted virion fraction again demonstrated the 3 major glycoproteins, and the 155K and 45K polypeptides. Comparison of structural polypeptides with the 16 constituents of the VZV-specific immunoprecipitation profile identified at least 1 polypeptide (145K) which was not represented in the virion and assumed to be nonstructural.