Comparison o;f vesicular stomatitis virus intracellular and virion ribonucleoproteins.
Vesicular stomatitis virus ribonucleoproteins (RNP) obtained by a detergent treatment of purified virus (vRNP) or from infected HeLa cell cytoplasm (icRNP) were examined by sedimentation in sucrose or Renografin gradients in the presence or absence of EDTA. It was shown that vRNP and icRNP sediment at the same rate in sucrose and Renografin in the absence of EDTA; however, icRNP sedimented more slowly in the presence of EDTA than did vRNP. Polyacrylamide gel electrophoresis of the proteins of vRNA and icRNP recovered from EDTA-containing gradients demonstrated that both RNP structures contained L, N, and NS proteins in the same proportion. Electron microscopy of both RNP structures, in the absence of EDTA, demonstrated that both exist as helical structures approximately 20 by 700 nm. However, in the presence of EDTA the icRNP was completely uncoiled with a mean length of 4,095 nm, whereas vRNP was hardly affected. The addition of excess Mg(2+) or Mn(2+) to uncoiled icRNP preparations partially restored the coiled configuration. These observations suggest that the change in sedimentation of icRNP in the presence of EDTA is due to a change from a coiled to an uncoiled conformation, that icRNP and vRNP are not structurally identical, and that icRNP must undergo a conformational change during maturation of VSV from the 20-by-700-nm intracellular form to the 50-by-175-nm form found in intact virus. The icRNP containing L, N, and NS proteins (icRNP(L,N,NS)) and icRNP containing only N protein (icRNP(N)), prepared by centrifugation of icRNP(L,N,NS) in CsCl to remove L and NS, were compared by cosedimentation in sucrose gradients. There was a decrease in sedimentation rate of icRNP(N) due to loss of L and NS. This sedimentation difference was also apparent in the presence of EDTA; however, both icRNP(L,N,NS) and icRNP(N) sedimented at a much slower rate in the presence of EDTA, and by electron microscopy both were completely uncoiled. These observations suggest that N protein alone is responsible for the 20-by-700-nm coiled structure and that the divalent cation interactions disrupted by EDTA are N-N or N-RNA interactions. These results are discussed with regard to vesicular stomatitis virus maturation.
D002413:Cations, Divalent / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D004492:Edetic Acid / Q000494:pharmacology; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D008854:Microscopy, Electron; D008968:Molecular Conformation; D009698:Nucleoproteins / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis; D012261:Ribonucleoproteins / Q000032:analysis*; D014764:Viral Proteins / Q000032:analysis; D014779:Virus Replication
D002413:Cations, Divalent; D009698:Nucleoproteins; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins; D004492:Edetic Acid