In this repot we review the evidence that synthesis and processing of Pr76 gag , the precursor to the core proteins (products of the gag gene) of avian retroviruses, is both necessary and sufficient to produce a virus particle. In addition, we describe the results of two-dimensional polyacrylamide gel electrophoretic analysis of Pr76 gag and its cleavage products. When the two-dimensional gel system is used in conjunction with protein blotting and antibody detection on nitrocellulose sheets, we observe multiply-charged species of both precursor and product proteins. Comparison with 32 PO 4 and 35 S-methionine labelled proteins indicates that much of the heterogeneity is probably due to phosphorylation. We also report results of an investigation into the site of synthesis of Pr76 gag . Immunoprecipitation across sucrose gradients containing polyribosomes derived from cytoplasmic and membrane fractions of infected cells indicates that Pr76 gag is found exclusively on cytoplasmic polyribosomes. Both Pr76 gag and, what we presume to be related incomplete nascent polypeptide chains, are localized in the 450S region of the gradient and are released by treatment with EDTA. Finally, we describe studies on a deletion mutant which, while failing to produce either reverse transcriptase or envelope glycoprotein, synthesizes pp60 src and a 63,000 dalton protein (P63) related to retrovirus core proteins. P63 contains an approximately 13,000 dalton internal deletion (in p27) as determined by tryptic mapping and analysis of products of in vitro cleavage by the gag-specific virion protease. The deletion does not appear to extend into the region of the p15 cleavage protease. P63 is resistant to normal processing and no product proteins are detectable even in cells superinfected with non-defective viruses. We conclude that the internal deletion in P63 has rendered this protein defective for processing either by inducing a conformational change, removal of a cleavage site, or both.

To learn more about the subcellular localization and nature of the enzymes involved in the post-translational processing of pro-ACTH/endorphin, separated rat anterior and intermediate pituitary tissue was homogenized and fractionated into subcellular organelles. Use of Percoll as a centrifugation material resulted in an excellent purification of secretory granules from pituitary tissue in a short period of time with very little handling. The purity of the granule and rough endoplasmic reticulum fractions was assessed by electron microscopy and by assays of marker enzymes for mitochondria (fumarase), rough endoplasmic reticulum (NADPH-cytochrome C reductase), Golgi (galactosyl transferase), and cytoplasm (lactate dehydrogenase). The amount of each form of ACTH and β-endorphin in the granule fraction, the rough endoplasmic reticulum/Golgi fraction and homogenate was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioimmunoassay. In both lobes of the pituitary the granule fractions were relatively enriched in the smaller peptide products derived from pro-ACTH/endorphin, while the rough endoplasmic reticulum/Golgi fractions were enriched in common precursor.

Bovine hypothalamic mRNA was translated in wheat germ and reticulocyte lysate cell-free systems.Translation products were immunoprecipitated with specific antisera and analyzed by SDS-PAGE.

Although it is generally accepted that all poliovirus specific proteins synthesized in infected cells are derived from one primary translation product, the poliovirus polyprotein (Jacobson & Baltimore, 1968), pulse labeling with 35 S-methionine of poliovirus infected cells under conditions of restricted polypeptide chain initiation leads to the accumulation of viral proteins in ratios different from those found under normal conditions. The accumulation of viral proteins coded for by the 5′ part of the viral mRNA (NCVP1a and its cleavage products) is twofold more inhibited by increased medium osmolarity than the accumulation of the other viral proteins notably NCVPx and NCVP1b and NCVP2. In addition, we have observed that the first virus specific proteins which appear during the replication cycle of polioviruses are NCVPx, NCVP1b and NCVP2. NCVP1a is only detectable at later times in infection. Therefore we propose that either NCVP1a is preferentially degraded early in infection or when cells are incubated in medium with increased osmolarity or that viral protein synthesis is initiated at more than one site. The presence of guanidine in the growth medium during the early part of the replication cycle results in dramatic alterations in the labeling pattern of poliovirus proteins indicating that guanidine affects the processing of viral proteins. Altered processing might be the cause for the inhibition of viral RNA synthesis.

The focus of research in our laboratory over the few years has been the regulation of synthesis, processing, and release of the ACTH/LPH family of peptides. These peptides are derived from a common precursor protein that is found in both the anterior and intermediate lobes of the pituitary (Roberts et al., 1978) and in the hypothalamus (Liotta et al., 1979). In the anterior lobe this protein gives rise to α (1–39) ACTH, β-lipotropin (β-LPH) and an N-terminal fragment of undefined function. In addition, a variety of intermediate lobe pituitary peptides can be derived from the precursor by further processing of ACTH and β-LPH. In this paper we compare the structure of the precursor in the anterior and intermediate lobes of mouse and rat pituitary. Processing of the precursor to its constituent hormones is then contrasted in primary cultures of anterior and intermediate lobe cells using pulse label and pulse chase techniques with radioactive amino acids and sugars.

MuLV Pr65 gag , the uncleaved polyprotein precursor to the internal group-specific antigens (gag) of murine leukemia viruses (NH 2 -p15-p12-p30-p10-COOH) is enriched for in immature particles. The immature particles are defined from thinsection electron micrographs as virions having a darkly-stained component apposed to the core shell interior and an electron-lucent center. Cleavage of Pr65 gag to the internal antigens, e.g., p30 and p12, is accompanied by a morphological change from an immature to a mature (uniformly electron-dense) form of the core. These in vitro events probably parallel those associated with budding and subsequent maturation of newly released virus particles. The proteolytic activity (MuLV-PF) responsible for these events is present in most virions harvested after 24 hours; those particles which lack the activity remain immature. The MuLV-PF activity in crude extracts has a pH optimum in the range of 7.0 to 7.4, requires 2% NP-40 or Triton X-100, and is inhibited by 0.10 M NaCl. Further, the partially purified MuLV-PF has a MW of about 20–22K, is a minor, highly unstable component of the virions and, based on inhibitor studies, appears similar to the serine protease, trypsin. In contrast to MuLV-PF, the avian tumor virus p15 (AMV-p15) associated protease is a major avian virion structural component which is stable in 0.05% SDS and appears to have a thiol-like protease activity. Our results suggest that there are at least two thiol protease-sensitive cleavage sites on Pr65 gag : one at the COOH terminus of p15 and the other near the p30-p10 junction. Taken together with earlier results showing that MuLVPF cleaves Pr65 gag at the p12-p30 junction to yield Pr40 gag , an intermediate product, we suggest, as have Oroszlan and Gilden (1980), there may be two protease activities responsible for cleavage of MuLV Pr65 gag .

The intermediate lobe of the frog and mouse pituitary synthesizes a glycoprotein precursor (pro-ACTH/αMSH-endorphin) which is processed to adrenocorticotropins (mol. wt. ˜21K, ˜13K and 4. 5K ACTH, K=1000 daltons), α-melanotropin (αMSH) and β-endorphin. The role of glycosylation of this precursor in the processing, packaging and secretion of its peptide products was examined using an inhibitor of glycosylation, tunicamycin. Pulse-chase experiments using the frog and mouse intermediate lobes showed that synthesis of the precursor was not affected by tunicamycin. However, in the presence of tunicamycin, the newly synthesized precursor underwent rapid intracellular degradation. Precursor molecules that were not completely degraded were processed to yield atypical peptides. These data suggest that the carbohydrate on the ACTH/αMSH-endorphin precursor may confer specific conformational properties, and direct its processing by limiting proteolysis. Subcellular fractionation and morphological studies on the frog intermediate lobe, in the presence of tunicamycin, show that glycosylation of pro-ACTH/ αMSH-endorphin was not essential for its packaging into secretory granules. Release of the atypical peptides processed from the unglycosylated precursor in tunicamycin treated lobes was also unaffected.