Studies on the intracellular distribution of Sindbis messenger RNA in infected chick-embryo fibroblasts. 1. Presence of extrapolyribosomal 26-S RNA in the membrane fraction.
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 polyribosomes 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.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D005347:Fibroblasts / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008854:Microscopy, Electron; D009154:Mutation; D009994:Osmolar Concentration; D011132:Polyribosomes / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D051381:Rats; D012845:Sindbis Virus / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012367:RNA, Viral; D011691:Puromycin