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The submicrosomal distribution of membrane-associated viral 26-S RNA in chick embryo fibroblasts infected with Sindbis virus was studied. Infected chick embryo fibroblasts were homogenized in the presence of low amounts of EDTA and fractionated by differential centrifugation. Analysis of postmitochondrial supernatants by isopycnic flotation on continuous sucrose gradients showed that membrane-associated 26-S RNA and ribosomes were not distributed in parallel, with an enrichment in 26-S RNA in the light microsomal subfractions. This distribution could not be explained by adsorption artifacts. Analysis of the distribution of microsomal constituents on sucrose gradients after treatment with digitonin ruled out the possibility that the 26-S RNA might be associated with Golgi or plasma membranes. The attachment of viral RNAs to endoplasmic reticulum membranes is discussed in relation to its possible role in viral morphogenesis. [ABSTRACT FROM AUTHOR]

Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron microscopy and cell fractionation. Electron microscopy of infected and non-infected cells revealed that infection produced a disaggregation of polyribosomes into monomers. Apart from this observation most cells appeared well preserved, and no degranulation of the rough endoplasmic reticulum was visible. Analysis of postnuclear supernatants by sucrose density gradients showed that no change in the relative proportions of free and membrane-bound ribosomes was produced by infection. Approximately 30 % of the ribosomes and 50 % of the viral RNA were found to be associated with membranes. Of the membrane-associated viral RNA, 70 % was recovered as 26-S RNA. Similar results were obtained with fibroblasts infected by the temperature-sensitive Sindbis mutant ts2, which is defective in the co-translational processing of the viral gene products at the nonpermissive temperature. Sucrose gradient analysis of membrane-bound polyribosomes solubilized by detergent indicated that as much as 50 % of the membrane-associated viral 26-S RNA is not integrated into potyribosomes and that most of the ribosomes are present as monomers or ribosomal subunits. Treatment with puromycin of living cells or of isolated membrane fractions under a variety of ionic conditions revealed that the viral RNA-membrane linkage is insensitive to puromycin but sensitive to high concentrations of monovalent ions. The bulk of the membrane-bound ribosomes were detached by high salt and recovered as ribosomal subunits on sucrose gradients. These results are consistent with the idea that in chick embryo fibroblasts infected with Sindbis virus only a small percentage of the ribosomes are engaged in protein synthesis, and that the Sindbis messenger RNA may attach to endoplasmic reticulum membranes through a ribosome-independent, salt-sensitive link. [ABSTRACT FROM AUTHOR]