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Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo fibroblasts.

ABSTRACT: In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma virus and from untransformed fibroblasts infected with a transformation-defective derivative of Prague C Rous-sarcoma virus, we have detected transformation-dependent alterations in both the acidic-type and the neutral-type asparagine-linked oligosaccharides of cellular glycoproteins. Pronase-digested glycopeptides were analysed by the combined techniques of gel filtration, exo- and endo-glycosidase digestion and concanavalin A-agarose affinity chromatography. The transformed cell glycoproteins contained more sialic acid and were enriched for more highly branched (versus biantennary) acidic-type structures compared with the untransformed cell glycoproteins, similarly to previously reported transformation-dependent alterations. In addition, the glycopeptides from the virus-transformed cells contained several neutral-type structures that were apparently absent from the untransformed cells: small neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to endo-beta-N-acetylglucosaminidase D but resistant to endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of 'truncated' precursor oligosaccharides (endoglycosidase-resistant, alpha-mannosidase-sensitive). Endoglycosidase-released oligosaccharides with the properties of hybrid-type structures were derived from the glycoproteins of both transformed and untransformed cells.

SUBMITTER: Hunt LA

PROVIDER: S-EPMC1145076 | biostudies-other | 1985 Jul

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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Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo fibroblasts.

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