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Studies of the decrease of tyrosine-O-sulphated proteins in Rous sarcoma-virus-transformed rat embryo fibroblasts, line 3Y1. Examination of the sulphate activation and tyrosyl-protein sulphotransferase systems.

ABSTRACT: The sulphate activation and tyrosyl-protein sulphotransferase systems in normal 3Y1 rat embryo fibroblasts and the same cells transformed by Schmidt Ruppin subgroup-A-Rous sarcoma virus (SRA-3Y1) were examined. Employing metabolic [35S]sulphate-labelling followed by PEI (polyethyleneimine)-cellulose thin-layer chromatography of the labelled cell lysates, it was found that the steady-state level of 'active' sulphate, adenosine 3'-phosphate 5'-phosphosulphate, was drastically lower in SRA-3Y1 cells compared with their normal counterparts. When the sulphate activating enzymes were tested, it appeared that the activities in 3Y1 homogenates were 2-2.5 times greater than those in SRA-3Y1 homogenates. An endogenous sulphation assay for tyrosyl-protein sulphotransferase revealed that activities in 3Y1 and SRA-3Y1 homogenates were comparable. Nearly identical patterns were observed with both sets of cells when [35S]sulphated proteins generated in the endogenous assay were separated by two-dimensional gel electrophoresis. It therefore seems that the tyrosyl-protein sulphotransferase(s) are unimpaired in SRA-3Y1 cells. While the lower (approx. 8 times) sulphate uptake remains the major cause for the decrease of tyrosine-O-sulphated proteins in SRA-3Y1 cells [Liu & Lipmann, (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3695-3698], the 2-2.5-fold lower sulphate activating enzyme activities also contribute to some extent to the difference between the SRA-3Y1 and 3Y1 cells.

SUBMITTER: Liu MC

PROVIDER: S-EPMC1148388 | biostudies-other | 1987 Oct

REPOSITORIES: biostudies-other

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Project description:UnlabelledPurified retroviral Gag proteins can assemble in vitro to form immature virus-like particles (VLPs). By cryoelectron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely, the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N and C termini, as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag compared that of assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly.ImportanceThe structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher-order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of antiretroviral drugs that interfere with virus assembly.

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Studies of the decrease of tyrosine-O-sulphated proteins in Rous sarcoma-virus-transformed rat embryo fibroblasts, line 3Y1. Examination of the sulphate activation and tyrosyl-protein sulphotransferase systems.

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