Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin

Abstract

Kunjin virus-infected cells were lysed and the cytoplasmic extract was subjected to sedimentation analysis. After centrifugation at 16,000 x g for 10 min about 70% of the original RNA-dependent RNA polymerase (RDRP) was recovered in the pellet; most of this enzymic activity was recovered in the soluble fraction after treatment with NP40 detergent. Membrane fractions were prepared from cytoplasmic extracts by centrifugation in discontinuous density gradients comprising w/w or w/v sucrose solutions, either for 3 h (top-loaded on 4 ml 20-60% sucrose) or for 19 h (centre-loaded in 37 ml 0-60% sucrose). Similar separations of bands of light membranes were obtained in all gradients. Multi-layered heavy membrane bands obtained with w/w sucrose gradients were resolved into two well-separated bands (F4 and F5) using w/v sucrose gradients. Thin-section electron microscopy of embedded membrane fractions, gel analysis of intracellular RNA, and RDRP assays showed that the w/w centre loading method and the w/v top-loading (short spin) method produced similar recoveries and distributions of smooth and rough membranes, intact virus particles and RDRP activity. The distribution of intracellular viral RNA and proteins was coincident with the RDRP, all being located in the F4 and F5 bands which contained the characteristic membrane structures induced during flavivirus infection. Significant advantages of the preferred method (w/v sucrose, top loading and short spin) were its rapidity, good preservation of membranes and RDRP, and the concentrations of RDRP achieved in the small volume fractions collected from a total of 4.5 ml.