Composition, arrangement and cleavage of the mouse mammary tumor virus polyprotein precursor Pr77gag and p110gag.

The amino acid sequence relationship between the nonglycosylated structural proteins of murine mammary tumor virus and the polyproteins from infected cells immunoprecipitated with an anti-p27 serum were examined using two-dimensional tryptic peptide mapping procedures. The proteins were labeled with ¹⁴C-lysine and ¹⁴C-arginine so that all but one of the tryptic peptides released from a protein could be detected. Previous studies have shown that immunoprecipitation of mammary tumor cells with anti-p27 serum results in the isolation of seven proteins in the molecular weight range of 34,000–160,000 daltons; and that cell-free translation using viral genomic RNA yields three p27-related proteins of 160,000, 110,000 and 77,000 daltons, similar to the three high molecular weight proteins detected in vivo. The proteins of lower molecular weight were thought to be cleavage intermediates of Pr77^gag. As judged from the peptide maps, Pr77^gag contained the complete sequences of the four major internal proteins of the virion (p27, pp21, p14 and p10) and possibly a fifth highly basic protein (p8) also found in virions. The putative cleavage intermediates, as expected, lacked some tryptic peptides that could be assigned to one or more of the major virion proteins and thus allow a scheme for the cleavage events to be constructed. p110^gag contained all the tryptic peptides found in Pr77^gag, plus some additional peptides. A minor virion protein p30 was found to include the peptides of p14 as well as some of the additional peptides present in p110^gag, suggesting a precursor-product relationship between the pr110^gag and p30. The data obtained from these studies lead us to propose that there are three protein precursors which include, at least in part, the gag gene region of the virion—p160 (potentially a gag/pol precursor), p110^gag and Pr77^gag—and that the arrangement of the virion proteins within the gag gene (pr77^gag) is p10-pp21-p27-p14.     

Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides.

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.     

Analysis of Immunoprecipitated Surface Glycoproteins in Measles Virions and in Membranes of Infected Cells

Measles viral envelope proteins were immune precipitated from membranes of infected cells and from purified virus and analyzed by polyacrylamide gel electrophoresis. Under reducing conditions, specific precipitates contained two major polypeptide bands, designated virus glycopeptides 1 and 2 (VGP-1 and VGP-2). Both polypeptides appeared to be glycosylated, as indicated by their incorporation of [¹⁴C]glucosamine in infected cells. VGP-2 appeared as a single band in specific precipitates of infected cells and as a double band in precipitates of purified virus. Trypsin treatment of infected cells showed that reduced VGP-2 may be composed of two unrelated polypeptides. One may be F₁, which is unglycosylated, and the other may correspond to the proteolytic cleavage product of VGP-1, which is glycosylated. The relation of VGP-1 and VGP-2 to smaller surface antigens (X and Y) obtained by tryptic treatment of infected cells remains to be elucidated. In cells taken at various times postinfection and analyzed for viral membrane proteins, VGP-1 was detected at all times, indicating that the input virus VGP-1 was inserted into the cell and could not be differentiated from newly synthesized VGP-1. VGP-2 was not detectable before 24 h postinfection. In precipitates of cells 4 h postinfection and of infected cells incubated at pH 5.8, an additional polypeptide band migrated immediately ahead of VGP-1. We conclude that VGP-2 (molecular weight, 42,000) possibly consists of two components, one of which is the tryptic cleavage product of VGP-1 and the other of which is the unglycosylated polypeptide, F₁.     

Processing of Adenovirus 2-Induced Proteins

Analysis of (35)S-methionine-labeled extracts of adenovirus 2-infected KB cells revealed 22 virus-induced polypeptide components. Most proteins of the virion were easily detected in extracts of whole cells labeled for short periods between 15 and 30 h after infection; however, several virion components were conspicuously absent. Radioactivity appeared in two of these virion components during a chase in nonradioactive medium, and this appearance was paralleled by a decrease in the radioactivity associated with two nonvirion adenovirus-induced proteins, results which imply precursor-product relationships for these components. Comparison of one of the chasable adenovirus-induced components (designated P-VII; mass of 20,000 daltons) and the major core protein (VII; mass of 18,500 daltons) of the virion showed that they have four common methionine-containing tryptic peptides; P-VII has an additional methionine residue which is not found in the major core protein. We propose that at least two of the adenovirus 2 virion components are derived by the cleavage of higher molecular weight precursor polypeptides.     

Immunological and Chemical Identification of Intracellular Forms of Adenovirus Type 2 Terminal Protein

Highly purified adenovirus type 2 terminal protein (TP) with an apparent M_r of 55,000 (55K) was prepared in quantities of 10 to 30 μg from guanidine hydrochloride- or sodium dodecyl sulfate-disrupted virions (60 to 120 mg). Guinea pigs were immunized with 14 to 20 injections of TP in amounts of 1 to 2 μg. Antiserum to TP was used to study the intracellular polypeptides related to adenovirus type 2 TP. By immunoprecipitation with anti-TP serum, we identified 80K and 76K polypeptides in the nucleoplasmic and cytoplasmic S100 fractions of [³⁵S]methionine-labeled cells early and late after infection with Ad2. By immunoautoradiographic analysis which eliminates coprecipitation of unrelated proteins, we identified an 80K polypeptide (probably an 80K-76K doublet) in unlabeled, late infected cells, using anti-TP serum and ¹²⁵I-labeled staphylococcal protein A. About two- to threefold-higher levels of the 80K and 76K polypeptides were present in the nucleoplasm than in the S100 fraction, and two- to threefold-higher levels were found in late infected cells than in early infected cells (cycloheximide enhanced, arabinofuranosylcytosine treated). We did not detect the 80K or 76K polypeptide in uninfected cells, indicating that these polypeptides are virus coded. Tryptic peptide map analysis showed that the 80K and 76K polypeptides are very closely related and that they share peptides with the DNA-bound 55K TP. Our data provide the first direct demonstration of intracellular 80K and 76K forms of TP. The intracellular 80K and 76K polypeptides are closely related or identical to the 80K polypeptide that Challberg and co-workers (Proc. Natl. Acad. Sci. U.S.A. 77:5105-5109, 1980) detected at the termini of adenovirus DNA synthesized in vitro and to the 87K polypeptide that Stillman and co-workers (Cell 23:497-508, 1981) translated in vitro. We did not detect the 55K TP in early or late infected cells, consistent with the proposal by Challberg and co-workers that the 80K polypeptide is a precursor to the virion-bound TP and that the conversion of the 80K polypeptide to the 55K TP occurs during virus maturation. The 80K and 76K polypeptides have many more methionine-containing tryptic peptides than does the 55K TP, and most of the tryptic peptides unique to the 80K and 76K polypeptides are very hydrophobic. Thus, the conversion of the 80K and 76K polypeptides to the 55K TP may involve the removal of a specific hydrophobic protein region.     

Defective interfering particles of poliovirus. II. Nature of the defect

Poliovirus defective, interfering particles in which about 15% of the standard viral RNA is deleted have been described (Cole et al., 1971). Stocks of DI³ particles more than 99% free of standard poliovirus were prepared by centrifugation of mixed preparations in CsCl gradients. Using purified DI particles, it was found that DI particles can carry out most of the standard poliovirus functions including inhibition of cellular macromolecular synthesis, production of viral RNA and production of virus-specific protein. Neither the kinetics nor extent of viral RNA or protein synthesis differed between DI particle-infected cells and standard virus-infected cells.Newly made virions, capsid proteins, and the capsid protein precursor (NCVP 1) were totally absent in DI particle-infected cells. All of the other viral proteins were present. DI-infected cells briefly labeled with amino acids also contained a new polypeptide, DI-P, which was apparently the residual fragment of NCVP 1 encoded by the DI genome. It was very unstable, being rapidly degraded to acid-soluble fragments. When the cleavage of viral proteins was inhibited with amino acid analogs, precursors of the viral proteins were generated. Those precursors which should have contained NCVP 1 had molecular weights 30,000 to 40,000 daltons lower in DI-infected cells than in standard virus-infected cells. This is the amount of protein encoded by 15% of the standard poliovirus genome which is the per cent of the standard RNA sequence not represented in DI RNA.Poliovirus DI particles therefore appear to be deletion mutants lacking RNA encoding about one-third of the capsid protein precursor. Whether the deletion is internal or terminal remains to be determined.     

Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins

Mouse mammary tumor virus (MMTV) derived from the culture medium of GR cells contained seven proteins, identified as gp55, gp33, p25, pp20, p16, p12, and p10. The major viral phosphoprotein was the 20,000-molecular-weight protein, pp20. Immunoprecipitation of cytoplasmic extracts from pulse-labeled GR cells identified three MMTV gag-specific proteins, termed Pr78(gag), Pr110(gag), and Pr180(gag+). These intracellular polyproteins were precipitable from cytoplasmic extracts by antisera to virions p25 and p12 but not by antisera to gp55. The major intracellular gag-specific precursor polyprotein, Pr78(gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, p10, and presumably pp20. This precursor is presumably derived from nascent chain cleavage or rapid posttranslational cleavage of the larger intracellular precursor-like protein, designated Pr110(gag). Pr110(gag) contained all but one of the leucine-containing tryptic peptides of Pr78(gag), plus several additional peptides. In addition to Pr78(gag) and Pr110(gag), monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weight precursor-like protein, designated Pr180(gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78(gag) and Pr110(gag) plus several additional peptides. By analogy to type C viral systems, Pr180(gag+) is presumed to represent a gag-pol common precursor which is the major pathway for synthesis of MMTV polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two env-specific proteins, designated gPr76(env) and gP79(env). The major env precursor, gPr76(env), could be labeled with radioactive glucosamine and was shown to contain antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A minor glycoprotein, gP79(env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79(env) represents fucosylated gPr76(env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.     

Synthesis of Sindbis virus nonstructural polypeptides in chicken embryo fibroblasts.

The identification of eight previously undescribed polypeptides in chicken embryo cells infected with Sindbis virus is reported. Seven of these polypeptides were distinguishable from the virus structural polypeptides and their precursors by their molecular weights and tryptic peptide maps. The eighth was closely related to pE2 (Schlesinger and Schlesinger, 1973), a precursor to one of the virus particle glycoproteins. Pulse-chase experiments and the use of an inhibitor of proteolytic cleavage allowed a division of the seven nonstructural (NS) polypeptides into three stable end products (NS p89, NS p82, and NS p60) and four precursors (p230, p215, p150, and p76). The labeling kinetics after synchronous initiation of translation indicated that synthesis of the NS polypeptides started at a single site and showed that the order of the genes coding for the NS polypeptides was (5' leads to 3') NS p60, NS p89, and NS p82. Short-pulse experiments under conditions of both synchronized and nonsynchronized translation suggested that cleavage of the primary translation product of the NS genes occurred only after its synthesis was completed and that the first cleavage removed the C-terminal polypeptide. From these and other experiments, we propose a detailed scheme for the synthesis and processing of Sindbis virus NS polypeptides.     

Rapid metabolism of moloney leukemia virus precursor polypeptides in virus infected Swiss mouse embryo cells.

Viral protein synthesis in Moloney murine leukemia virus infected high passage mouse embryo cells was studied utilizing monospecific antisera to the viral core protein p30 and envelope protein gp71. Pulse-chase analysis of [³⁵S]methionine-labeled polypeptides in combination with the demonstration of the presence of either gp71 or p30-specific antigenic determinants in them indicated a 84,000-dalton polypeptide as the precursor of viral glycoproteins and four metabolically unstable polypeptides of approximate molecular weights 88,000, 72,000, 62,000, and 39,000 as the precursors of viral core protein, p30. The p30-containing 88,000 and 72,000-dalton polypeptides were distinctly seen in this system under normal growth conditions. Further, the processing of p30 precursors was very rapid and was complete during a 40 min chase while only partial processing of glycoprotein precursor was observed during the same period.     

Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao

The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M_r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A)⁺RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A absorbance - cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies.     

Characterization of the Polypeptides Formed in Response to Encephalomyocarditis Virus Ribonucleic Acid in a Cell-Free System from Mouse Ascites Tumor Cells

The polypeptide products synthesized at different times in a cell-free system from Krebs mouse ascites tumor cells in response to the addition of encephalomyocarditis (EMC) virus ribonucleic acid (RNA) were characterized by electrophoresis on polyacrylamide gels and fingerprint analysis of their tryptic peptides. Translation of the EMC RNA genome with time occurred in a nonrandom fashion in these systems, to yield products containing sequences characteristic of both virion capsid polypeptides and EMC-specific polypeptides present only in the infected cell. The molecular weights of the products fell in a series from 20,000 to 140,000 daltons, although occasionally traces of larger polypeptides were also observed. All of the major polypeptides appeared to arise from partial or complete translation of about 60% of the EMC RNA genome. They were not formed by cleavage of a large precursor molecule. It is suggested that they are artifacts generated by premature "termination" of nascent polypeptide chains at preferred sites.     

Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors.

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.