Purification of herpes simplex virus type 1 by density gradient centrifugation and estimation of the sedimentation coefficient of the virion.
Purification of herpes simplex virus type 1 (HSV-1) is often performed by centrifugation in gradients of various materials. A major problem with such procedures is a gradual decrease in the infectivity of the virus, probably due to the influence of the gradient material. In the present work we have compared Nycodenz gradients with Ficoll gradients for HSV-1 purification. Both gradient materials have low osmolarities. The purity of the preparations recovered from the two gradients was similar, as measured by electron microscopy and two-dimensional gel electrophoresis, and the yield of infectious virus was approximately the same. Szilágyi & Cunningham (22) reported previously that Ficoll gradients separate one band of complete virions and another band containing light particles devoid of nucleocapsids. In Nycodenz gradients we observed the same bands, and an additional third band which contained aggregates and disrupted particles. In this respect, the separation in Nycodenz is slightly superior to that in Ficoll. The purified HSV-1 virions were subjected to analytical differential centrifugation and an S-value of 670S was calculated for the virus.
Sathananthan
B
B
Centre for Research in Virology, University of Bergen, Bergen High Technology Centre, Norway.
Rødahl
E
E
Flatmark
T
T
Langeland
N
N
Haarr
L
L
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D005362:Ficoll; D018259:Herpesvirus 1, Human / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure*; D007472:Iohexol; D007668:Kidney; D008715:Methionine / Q000378:metabolism; D008854:Microscopy, Electron; D014764:Viral Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014771:Virion / Q000254:growth & development / Q000302:isolation & purification / Q000648:ultrastructure
D014764:Viral Proteins; D005362:Ficoll; D007472:Iohexol; D008715:Methionine