Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C.

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid.

Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells.

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant.

Asparagine-linked glycosylation of the scrapie and cellular prion proteins.

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Inhibition of avian retrovirus protein synthesis in the presence of host cellular mRNA.

Two conditions of in vitro translation are described in which the RNA of Rous sarcoma virus is significantly less efficient at protein synthesis than host cellular chick mRNA. In direct competition, addition of increasing amounts of host cell mRNA inhibits synthesis of gag gene products from RSV RNA. When the denaturant dimethyl sulfoxide is added to a mixture of the two RNA's the inhibition is even greater.

Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins.

Scrapie and Creutzfeldt-Jakob disease are transmissible, degenerative neurological diseases caused by prions. Considerable evidence argues that prions contain protease-resistant sialoglycoproteins, designated PrPSc, encoded by a cellular gene. The prion protein (PrP) gene also encodes a normal cellular protein designated PrPC.

Identification of an initial site of interaction and possible helix destabilizing activity preceding initiation of protein synthesis from retrovirus RNA.

Hybrid arrest of translation initiation (HARTI) assays with a series of oligodeoxyribonucleotides demonstrated that in rabbit reticulocyte lysate (RRL), the initial binding of protein synthesizing components to Rous sarcoma virus RNA requires only the penultimate guanosine and cap structure. Assays of DNA:RNA heteroduplex stability in RRL suggest that helix destabilizing activities then play a role in subsequent initiation of protein synthesis.

Scrapie prion protein contains a phosphatidylinositol glycolipid.

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30.

Influence of base-pairing in the leader region on in vitro translation of Rous sarcoma virus RNA.

The capacity of the leader region of Rous sarcoma virus (RSV) RNA to act as a regulator of viral protein synthesis was tested in vitro. When DNA/RNA hybrids of sufficient length (greater than 90-100 nucleotides) are created within the leader, synthesis of Pr76gag is inhibited. The inhibition is dependent upon the length of the hybrid rather than its position with the exception that encumberance of the 5'-terminal 33 nucleotides does block translation.

Participation of 5'-terminal leader sequences in in vitro translation of Rous sarcoma virus RNA.

The cell-free translation of genome RNA from Rous sarcoma virus was examined following hybridization to selected fragments of viral DNA. Single-stranded fragments, generated by t-RNATrp primed transcription of 70 S RNA from the Schmidt-Ruppin D strain, were isolated and purified by electrophoresis. These included DNA complementary to the 5'-terminal 101 nucleotides (DNA100) of virion 38 S RNA and a collection of prematurely terminated transcripts which lack the complement to the extreme 5'-terminal 7-20 nucleotides (DNA less than 100).