Chemical and enzymatic characteristics of cytoplasmic membranes of poliovirus-infected HeLa cells

Abstract

The density distribution of cytoplasmic membranes separated by centrifugation to equilibrium in discontinuous sucrose gradients is progressively altered after infection of HeLa cells with poliovirus. The most striking changes are a decrease in quantity and an increase in density of rough microsomes and a large increase in quantity of the smooth microsomal membranes with which viral RNA synthesis is associated. The smooth membranes begin to increase between 2.5 and 3.5 hr after infection, and continue to proliferate late into infection. They differ from the membranes in the corresponding fraction of uninfected cells in their higher phospholipid:protein ratio, altered phospholipid composition, and reduced specific activity of NADH diaphorase. The specific activities of several enzymes were determined in membrane fractions obtained from discontinuous sucrose gradients. Mitochondrial and lysosomal enzyme activities are found mainly in a fraction which also contains most of the rough endoplasmic reticulum. However, 4 hr after infection a lysosomal enzyme, β-glucuronidase, is distributed throughout the gradient. Most of the plasma membrane fragments are sedimented in the low speed centrifugation prior to layering into the discontinuous sucrose gradients. The small proportion of the plasma membrane fragments that are layered into the gradients cosediment with the smooth microsomal membranes.