Ralph E. Smith

Polypeptide components of virions, top component and cores of reovirus type 3

Abstract The capsid protein moiety of virions of the Dearing strain of reovirus type 3 consists of seven species of polypeptides as determined by polyacrylamide gel electrophoresis. The molecular weights of these polypeptides have been estimated by comparing their rate of migration in SDS-containing gels with that of marker proteins of known molecular weights. The number of molecules of each polypeptide in the virion has also been determined. There are 113 and 80 molecules, respectively, of polypeptides λ 1 (M.W. 155,000) and λ 2 (M.W. 140,000); 23 and 550 molecules, respectively, of polypeptides μ 1 (M.W. 80,000) and μ 2 (M.W. 72,000); and 31, 202, and 890 molecules, respectively, of polypeptides σ 1 (M.W. 42,000), σ 2 (M.W. 38,000), and σ 3 (M.W. 34,000). In addition virions also contain about 500 molecules of a very small polypeptide, the molecular weight of which is 5000–10,000. The polypeptides in reovirus top component (particles lacking RNA) are identical with those of virions. Treatment with chymotrypsin or trypsin converts virions to cores which lack the outer shell of capsomeres. Cores contain capsid polypeptides λ 1 , λ 2 , and σ 2 . Cores can similarly be prepared from top component. Top component cores contain the same polypeptides as virion cores, but the enzyme treatment necessary for the conversion cleaves polypeptide λ 1 . The molecular weights of the three size classes of capsid polypeptides are such that they are probably coded by monocistronic messenger RNA molecules transcribed from the L, M, and S segments of reovirus genome RNA. The capsid polypeptides thus use the information in 2 of the 3 L segments, 2 (or possibly only 1) of the 3 M segments, and 3 of the 4 S segments.

Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses

Abstract Studies were designed to detect changes in the plasma membrane polypeptide composition of chick embryo fibroblasts (CEF) and Normal rat kidney (NRK) cells following infection with avian RNA tumor viruses. A method capable of isolating large fragments of plasma cell membranes, based on fractionation in a two-phase polymer system, was developed for this purpose. Infection with the Prague (subgroup C) or SR (subgroup A) strains of RSV, or with ts 68 of SR-RSV-A or ts 339 of B77 at the permissive temperature, caused CEF to transform morphologically; simultaneously a limited number of changes occurred in their plasma cell membrane composition. They were: (a) a large increase in the rate of labeling with 14 C-valine and in the total amount of a polypeptide with an apparent MW of 73,000; a similar small increase occurred with respect to a polypeptide with an MW of 95,000; neither could be labeled with 14 C-glucosamine; (b) a decrease in the rate of labeling and in the total amount of high molecular weight glycopoly-peptides with MWs of about 250,000; (c) a decrease in the rate of labeling and in the total amount of a polypeptide with an MW of 39,000. Infection with ts 68 and ts 339 at the nonpermissive temperature, or with a nontransforming mutant of SR-RSV-A, or with RAV-7, did not cause alterations (a) and (b), but did cause alteration (c). Identical membrane polypeptide changes were observed in NRK cells infected with the Prague strain of RSV or with ts 339 of B77 at the permissive but not at the nonpermissive temperature. The significance of the fact that these changes, in particular the increases in the amounts of the 73,000 and 95,000 dalton polypeptides, occurred to the same extent in the membranes of both avian and mammalian cells is discussed.

Comparison of immature (rapid harvest) and mature Rous sarcoma virus particles

Abstract RSV-Prague was harvested from transformed chick embryo fibroblasts 5, 10, 20, 60, and 180 min after release and compared with virus harvested after 12 and 24 hr with respect to the nature of its RNA, polypeptide constitution, and ability to use its own RNA and added DNA as primer/template for DNA synthesis. The following results were obtained: 1. a. Immature (5 min) virus contained little 68S RNA. The principal component of the native RNA of such virus was rather heterogeneous, with a median sedimentation coefficient of 55–60S; in addition, several other components were also present. In the denatured state immature virus RNA consisted of approximately equal parts of very homogeneous 36S RNA, RNA which sedimented between 36S and 4S, and 4S RNA. By contrast, the native RNA of mature (24 hr) virus consisted mostly of a homogeneous 68S component, which on denaturation yielded a heterogeneous population of molecules sedimenting between 36S and 4S. The RNA of virus at intermediate stages of maturity yielded profiles intermediate between these two patterns. 2. b. Immature (5 min) virus particles contained 26 readily identifiable polypeptides; mature (24 hr) virus particles contained only 13. Virus particles of intermediate age contained intermediate numbers of polypeptides. No precursor—product relationships were discernible; rather, it appeared that 13 polypeptides were lost during maturation and aging. 3. c. The ability of immature (5 min) virus particles to synthesize DNA was not enhanced by the addition of detergent (Triton X-100); on the other hand, the addition of calf thymus DNA as exogenous primer/template profoundly stimulated DNA synthesis. The reverse was true for mature 24-hr virus. This suggested that the location of the enzyme(s) responsible for DNA synthesis differed in immature and mature virus particles, and implied that the RNA of immature virus particles might not be in the proper configuration necessary for serving as a primer/template for DNA synthesis.

Immunological properties of avian oncornavirus polypeptides

Abstract The major polypeptides and glycoproteins of avian myeloblastosis virus and of the Prague strain of Rous sarcoma virus were isolated by gel filtration in guanidine hydrochloride (GuHCl). Hyperimmune sera prepared in rabbits against homogeneous preparations of each material were used to study the nature of the antigenic specificities on these molecules. The results indicated that (1) each component contained unique antigenic determinants; (2) each of four polypeptides (27,000, 19,000, 15,000, 12,000 daltons) contained group-specific (gs) reactivity; and (3) subgroup specific reactivity was found in the 19,000-dalton polypeptide.

Induction of anemia by avian leukosis viruses of five subgroups

Abstract Chickens infected with avian leukosis viruses frequently manifest anemia during the development of disease. Previous work has established that infection of the 8-day-old hatched chick with virus results in the synchronous appearance of a profound anemia. In this communication, we have used this phenomen to test viruses representing five subgroups of avian leukosis for the ability to induce anemia. The results indicate that viruses of B and D subgroups induced severe anemia while viruses of subgroups A, C, and F induced only mild anemia or none at all.

Evidence for common nucleotide sequences in the RNA subunits comprising Rous sarcoma virus 70 S RNA

Abstract Digestion of Rous sarcoma virus 32 P-labeled high molecular-weight RNA with T 1 ribonuclease resulted in the release of approximately 30 unique large oligonucleotides which were resolved by electrophoresis and chromatography on thin layers of cellulose and DEAE-cellulose. Determination of the radioactivity present in each oligonucleotide of known chain length and in the remaining digestion products made it possible to estimate the total number of residues expected in a polynucleotide chain containing the sequences represented. The oligonucleotides are present in amounts greater than would be expected for a unique and nonrepetitive 70 S RNA molecule with a molecular weight of 1 × 10 7 . Many of the sequences must, therefore, be present in more than one of the 35 S RNA subunits comprising the 70 S RNA.

The influence of carbohydrate on the antigenicity of the envelope glycoprotein of avian myeloblastosis virus and B77 avian sarcoma virus

The immunological reactivity of the surface glycoproteins (gp85) of avian myeloblastosis virus (AMV) and B77 avian sarcoma virus were examined by competition radioimmunoassay. The influence of the protein and carbohydrate portions of the glycoprotein on the antigenic reactivity was investigated by systematic digestions of AMV gp85 with proteases and glycosidases. Digestion of AMV gp85 with trypsin, trypsin and chymotrypsin, or Pronase had no effect on the antigenicity detected in competition radioimmunoassay using rabbit antibody made against AMV gp85. However, digestion of AMV gp85 with a mixture of highly purified glycosidases (neuraminidase, β-galactosidase, β-N-acetylglucosaminidase, and endo-β-N-acetylglucosaminidase) eliminated the reactivity detected in competition radioimmunoassay with the same antisera. The contribution of individual carbohydrates to the reactivity was studied by a sequential digestion of AMV gp85 with various combinations of glycosidases. These studies suggested that removal of sialic residues had no effect on antigenicity and that removal of β-galactose and β-N-acetylglucosamine residues abolished antigenic reactivity. In addition, glycosidase digestion of 125I-labeled AMV gp85 resulted in a decrease of reactivity in direct radioimmunoassay. These results indicate that the immunological reactivity of AMV gp85 depends upon an intact carbohydrate side chain. Digestion of AMV gp85 with proteases and glycosidases, followed by competition radioimmunoassay using antibody prepared against B77 gp85, showed that protease treatment reeulted in a partial loss of antigenic reactivity. Treatment of B77 gp85 with proteases and glycosidases, followed by competition radioimmunoassay using antibody prepared against B77 gp85, showed that protease treatment resulted in a partial loss of antigenic reactivity, while glycosidase treatment resulted in no loss of antigenic reactivity.

Viral glycoprotein synthesis under conditions of glucosamine block in cells transformed by avian sarcoma viruses

Abstract The synthesis of the viral glycoprotein gp85 in B77-transformed chick embryo cells cultured under conditions of glucosamine block was studied. As determined by long-term labeling with radioactive fucose, no gp85 is formed in the presence of 20 μmoles/ml of glucosamine, although an intracellular component was detected which was recognized by a serum prepared against gp85 but migrated in SDS-polyacrylamide gel with a greater mobility than gp85. The labeling properties of this component suggest that it is a carbohydrate-deficient form of gp85. These data are discussed in terms of a chemical mechanism for glucosamine block.

Collagen biochemistry of osteopetrotic bone: I. Quantitative changes in bone collagen cross-links in virus-induced avian osteopetrosis

Abstract The patterns of [3H]-NaBH4-reduced bone collagen cross-links from osteopetrotic chickens were compared with those of age-matched controls. Ratios of the reduced cross-links, dihydroxylysinonorleucine (DHLNL) 1 to hydroxylysinonorleucine (HLNL), were dramatically increased in tibia bone samples from osteopetrotic birds compared to values from control birds. In addition, the initial HLNL peak from osteopetrotic bone collagen was heterogeneous, whereas DHLNL from osteopetrotic or normal bone collagen and HLNL from normal bone collagen were homogeneous.

Identification of plaque isolates of an avian retrovirus causing rapid and slow onset osteopetrosis

This study examines the basis for the observation that serum stocks of myeloblastosis-associated virus MAV-2(0) obtained from chickens with osteopetrosis induced abnormal bone growth after a latent period of less than 3 weeks, while stocks of plaque-purified MAV-2(0) induced abnormal bone growth only after latent periods of 6 weeks or greater. To establish the basis for this distinction, 85 plaque isolates were derived from a serum stock of MAV-2(0), and each plaque isolate was injected into 10-day-old chick embryos. Infected chicks were examined 3 weeks after hatch for the weight of their body, bursa, and bone. Three MAV-2(0) plaque isolates caused bone growth that was at least fivefold greater in infected than in uninfected animals. The other 82 plaque isolates caused slight osteopetrosis or none at all. The serum stock of MAV-2(0) therefore consisted of two forms of virus: one caused rapid onset osteopetrosis, and the other caused slow onset osteopetrosis. Almost all plaque isolates of MAV-2(0) caused decreased body and bursa weights, but with the rapid onset plaque isolates, these effects were more severe. The ability to cause rapid onset osteopetrosis was stable for three sequential plaque purifications. RNA from rapid and slow MAV-2(0) plaque isolates did not differ in T1 oligonucleotide fingerprints, and all had RNA 8.2 Kb in length. Structural polypeptide compositions of rapid and slow plaque isolates were essentially the same, as defined by migration on SDS-polyacrylamide gels. The growth rate of the isolates on embryonic limb bud cells and chick embryo fibroblasts were similar.