Protein Composition of Chilo Iridescent Virus
Previous studies on interactions between Chilo iridescent virus (CIV) and cells have shown that several specific biological activities are associated with structural components of the virus particles. This is the case for enzymatic activities (e.g. ATPase, protein kinase and DNAse), cell fusion induction, inhibitory activity of cellular macromolecular syntheses and non-genetic reactivation of CIV DNA infectivity. In order to gain insight into viral pathogenesis, precise localization of the CIV polypeptides within the virion and characterization of their related biological properties is indispensable. This was achieved by different techniques: (i) analysis of CIV polypeptides cross-linked by disulfide bridges in two-dimensional gel electrophoresis, (ii) stepwise disruption and stripping of virus particles, (iii) detection of external polypeptides by surface labeling, (iv) detection of proteins involved in nucleoprotein complexes and (v) selective solubilization and reconstitution of the inner viral membrane.
Thus far, up to 43 polypeptides have been identified by two-dimensional SDS-polyacrylamide gel electrophoresis. In contrast with previous reports, the major capsid component was found to be constituted of two proteins, a protein of 150,000 daltons (150 kDa, P150) composed of three identical disulfide-linked polypeptide subunits of 50 kDa (P’50) and a 50 kDa protein (P50). Although differing in some of their properties, these two proteins showed identical migration in SDS-2-ME-PAGE, however they differed in some properties. The accessibility of their sulfhydryl residues was different and radioiodination of the external polypeptides showed only one single intensively labeled spot, corresponding to the P50. However, tryptic peptide analysis showed that P50 and P’50 had a very similar peptide composition. These observations indicated that the trimeric protein P150 was not directly exposed at the surface of the viral particle. In addition, P’50 did not seem to have any affinity for viral DNA.
Thermodynamic analysis of CIV particles suggested that the DNA would be packed into a nucleosome-like structure with a viral core composed of at least six DNA-associated polypeptides. Among the six DNA-binding proteins P12.5 represents the major constituent and the preferred substrate for the virus-coded protein kinase. Preliminary experiments suggested that phosphorylation of P12.5 may result in decondensation of the DNA molecule and contribute to the release of DNA from the viral nucleocapsid at early stages of infection.
Viral inner membrane can be readily solubilized with octylglucoside. Biochemical analysis of the vesicles obtained after action of the detergent, showed that only a few polypeptides were solubilized under these conditions. However, this soluble fraction was found to be of major importance in the first stages of virus infection. The solubilized fraction contained all the factors required to initiate the replication cycle. (i) The so-called “virus-promoting factor” (VPF) induced a full cell-fusion activity and this phenomenon is independent of the capsid virus proteins, (ii) The soluble fraction also contained an inhibitory protein that facilitated viral infection by switching off cellular macromolecular syntheses and (iii) a viral-activation protein factor that stimulated the transcription of immediate early genes by a cellular polymerase.
KeywordsCellulose Sucrose Urea DMSO Proline
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