Amino acid sequence of p15 from avian myeloblastosis virus complex

The complete amino acid sequence of the p15 gag protein from avian myeloblastosis virus (AMV) complex has been determined by sequential Edman degradation of the intact molecule and of peptide fragments generated by limited tryptic cleavage, cleavage with staphylococcal protease, and cyanogen bromide cleavage. AMV p15 is a single-chain protein containing 124 amino acids. The charged amino acids tend to be clustered in the primary structure. p15 contains a single cysteine at position 113 which may be essential for the p15 associated proteolytic activity. However, p15 shows no appreciable sequence homology with papain or other classical thiol proteases.     

Purification of viral proteins from avian sarcoma virus QV2.

A procedure was established whereby most of the major viral proteins were isolated to apparent homogeneity in biologically and immunologically active forms from a single batch of avian sarcoma virus QV2. For the initial step of purification, gently disrupted virions were fractionated by CsCl centrifugation into envelope proteins, RNA-dependent DNA polymerase, and viral core proteins. Further purification of envelope glycoproteins and DNA polymerase was performed by affinity chromatography on agarose columns cross-linked with plant lectins and poly(C), respectively. On the other hand, core proteins were fractionated by a combination of gel filtration and ion-exchange column chromatography into components p27, p19, and p15. The core protein p15 thus isolated retained proteolytic activity even after storage for 6 months. The present study also demonstrated that QV2 p19 is structurally altered from the corresponding protein of avian myeloblastosis virus (AMV), a reference avian leukosis-sarcoma virus having a well-characterized polypeptide composition.     

Amino-terminal amino acid sequence of p10, the fifth major gag polypeptide of avian sarcoma and leukemia viruses.

We have identified p10 as a fifth gag protein of avian sarcoma and leukemia viruses. Amino-terminal protein sequencing of this polypeptide purified from the Prague C strain of Rous sarcoma virus and from avian myeloblastosis virus implies that it is encoded within a stretch of 64 amino acid residues between p19 and p27 on the gag precursor polypeptide. For p10 from the Prague C strain of Rous sarcoma virus the first 30 residues were found to be identical with the predicted amino acid sequence from the Prague C strain of Rous sarcoma virus DNA sequence, whereas for p10 from avian myeloblastosis virus the protein sequence for the same region showed two amino acid substitutions. Amino acid composition data indicate that there are no gross composition changes beyond the region sequenced. The amino terminus of p10 is located two amino acid residues past the carboxy terminus of p19, whereas its carboxy terminus probably is located immediately adjacent to the first amino acid residue of p27.     

Primary structure of p19 species of avian sarcoma and leukemia viruses.

The internal structural proteins of avian sarcoma and leukemia viruses are derived from a precursor polypeptide that is the product of the viral gag gene. The N-terminal domain of the precursor gives rise to p19, a protein that interacts with the lipid envelope of the virus and that may also interact with viral RNA. The C terminus of p19 from the Prague C strain of Rous sarcoma virus was previously assigned to a tyrosine residue 175 amino acids from the N terminus. We have used metabolic labeling and carboxypeptidase digestion to show that the C terminus of p19 is actually tyrosine 155. This implies the existence of a sixth gag protein 22 amino acids in length and located between p19 and p10 on the gag precursor. The p19 species of some recombinant avian sarcoma viruses and of the defective endogenous virus derived from the ev-1 locus migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as if they were about 4,000 daltons smaller than p19. We have elucidated the structure of these forms, called p19 beta, by analysis of the proteins and determination of the DNA sequence of the p19 region of the gag gene from ev-1 and ev-2. Esterification of carboxyl groups completely suppressed the differences in migration of p19 and p19 beta. Peptide mapping showed the altered mobility to be determined by sequences in the C-terminal cyanogen bromide fragment of the proteins. We conclude from the DNA sequence that a single glutamate-lysine alteration is responsible for the altered electrophoretic mobility.     

Fractionation of two protein kinases from avian myeloblastosis virus and characterization of the protein kinase activity preferring basic phosphoacceptor proteins.

Two protein kinase activities were fractionated from purified virions of avian myeloblastosis virus. Distinguishing characteristics of these two protein kinases included: (i) their binding properties during purification by ion-exchange chromatography; (ii) their estimated molecular weights; and (iii) their phosphoacceptor protein specificities. The protein kinase that bound to the anion exchanger DEAE-cellulose (pH 7.2) had an estimated molecular weight of 60,000 to 64,000 and preferred basic phosphoacceptor proteins. The protein kinase that bound to the cation exchanger phosphocellulose (pH 7.2) had an estimated molecular weight of 42,000 to 46,000 and preferred acidic phosphoacceptor proteins. The protein kinase preferring basic phosphoacceptor proteins was further purified and characterized. Optimal transfer of phosphate catalyzed by this enzyme required a divalent metal ion, a sulfhydryl-reducing agent, and ATP as phosphate donor. GTP was not an effective phosphate donor at concentrations comparable to ATP; and the cyclic nucleotides cyclic AMP and cyclic GMP neither stimulated nor inhibited protein phosphorylation by the protein kinase. The specificity of the protein kinase for basic phosphoacceptor proteins extended to proteins from avian myeloblastosis virus, in that the neutral to basic virion proteins p12, p19, and p27 served as phosphate acceptors. In addition, the protein kinase also appeared to phosphorylate itself. The role(s) of this virion-associated protein kinase is discussed.     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. II. Cyanogen bromide peptides

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).     

Purification of Avian Myeloblastosis Virus DNA Polymerase by Affinity Chromatography on Polycytidylate-Agarose

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.     

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.     

Correlation of RNA binding affinity of avian oncornavirus p19 proteins with the extent of processing of virus genome RNA in cells.

We purified the p19 proteins from the Prague C strain of Rous sarcoma virus, avian myeloblastosis virus, B77 sarcoma virus, myeloblastosis-associated virus-2(0), and PR-E 95-C virus and measured their binding affinities for 60S viral RNA by the nitrocellulose filter binding technique. The apparent association constants of the p19 proteins from Rous sarcoma virus Prague C, avian myeloblastosis virus, and B77 sarcoma virus for homologous and heterologous 60S RNAs were similar (1.5 x 10(11) to 2.6 x 10(11) liters/mol), whereas those of myeloblastosis-associated virus-2(0) and PR-E 95-C virus were 10-fold lower. The sizes and relative amounts of the virus-specific polyadenylic acid-containing RNAs in the cytoplasms of cells infected with Rous sarcoma virus Prague C, myeloblastosis-associated virus-2(0), and PR-E 95-C virus were determined by fractionating the RNAs on agarose gels containing methylmercury hydroxide, transferring them to diazobenzyloxymethyl paper and hybridizing them to a 70-nucleotide complementary DNA probe. In cells infected with Rous sarcoma virus Prague C we detected 3.4 x 10(6)-, 1.9 x 10(6)-, and 1.1 x 10(6)-dalton RNAs, in PR-E 95-C virus-infected cells we detected 3.4 x 10(6)-, 1.9 x 10(6)- and 0.7 x 10(6)-dalton RNAs, and in cells infected with myeloblastosis-associated virus-2(0) we detected 3 x 10(6)- and 1.3 x 10(6)-dalton RNAs. Each of these RNA species contained RNA sequences derived from the 5' terminus of genome-length RNA, as evidenced by hybridization with the 5' 70-nucleotide complementary DNA. The ratios of subgenomic mRNA's to genome-length RNAs in cells infected with myeloblastosis-associated virus-2(0) and PR-E 95-C virus were three- to five-fold higher than the ratio in cells infected with Rous sarcoma virus Prague C. These results suggest that more processing of viral RNA in infected cells is correlated with lower binding affinities of the p19 protein for viral RNA, and they are consistent with the hypothesis that the p19 protein controls processing of viral RNA in cells.     

Monoclonal antibody against the carboxy terminal peptide of pp60src of Rous sarcoma virus reacts with native pp60src.

A monoclonal mouse antibody has been prepared against a synthetic peptide corresponding to the six carboxy-terminal amino acids (C' peptide) of the src gene product pp60v -src of Rous sarcoma virus (RSV). The antibody was able to precipitate pp60v -src and to bind pp60v -src kinase activity in a competition test, indicating that this peptide can serve as an antibody-binding site (epitope). Furthermore, the finding that three out of 28 pp60src-specific tumor-bearing rabbit (TBR) sera contained antibody against the C' peptide argues for an in vivo role for the carboxy terminus of pp60src. C' peptide-specific IgG was purified from one TBR serum using affinity chromatography, and was shown to precipitate significant amounts of pp60src, and bind most of the pp60src kinase activity from SRA, PrA, and B77-C strains of avian sarcoma virus (ASV), but not endogenous pp60c -src, a cellular homologue to the viral pp60v -src. Similar results were obtained with IgG isolated from a C' peptide immune rabbit serum. None of the three C' peptide-specific IgGs could serve as a phosphate acceptor in an immune complex protein kinase reaction.     

Differential proteolytic processing leads to multiple forms of the CA protein in avian sarcoma and leukemia viruses.

The CA (capsid) protein of avian sarcoma and leukemia viruses occurs in multiple species. Only one form has been previously characterized biochemically. We have now determined that the mature CA protein of avian sarcoma and leukemia viruses exists as three species with different C termini, ending in amino acid residues A-476, A-478, and M-479 of the Gag precursor, respectively. These structures were deduced from a combination of cyanogen bromide peptide mapping, sequence analysis of tryptic peptides, and electrospray mass spectrometry. The three forms of CA were detected in the same ratios in Rous sarcoma virus and avian myeloblastosis virus and therefore are likely to represent a common feature of members of this genus of avian retroviruses. The only previously reported CA species, CAM-479, accounts for only about 36% of the total CA protein, while CAA-476 and CAA-478 account for 55 and 9%, respectively. From the analysis of peptides cleaved in vitro by PR, the viral protease, we infer that the cleavage site between A-476 and A-477 not only is recognized by PR but is the preferred site. We were unable to determine if A-478/A-479 is a cleavage site for PR or alternatively if CAA-478 results from further processing of CAM-479 by a carboxypeptidase. To study the biological significance of residues A-477 to M-479, we constructed genetically altered viruses in which deletions removed either residues 477 to 479 or 477 to 488. The resulting virus particles appeared to assembly with normal efficiencies, but the latter mutant showed slowed proteolytic processing. Neither of the mutants was infectious.     

重组鲑鱼降钙素前体多肽的制备及其性质研究

硫氧还原蛋白的第 38位 Met突变为 Ala的 Trx- s CT- Gly基因在 E.coli BL2 1 ( DE3)中得到高效表达 .用 Thio Bind亲和树脂纯化表达的融合蛋白 .结果说明 38位的 Met突变为 Ala不影响融合蛋白与树脂的特异性结合 ,融合蛋白的纯度达 90 %以上 .在含有 3mol/L尿素的 70 %甲酸中 ,室温 48h,至少 80 %融合蛋白被溴化氰裂解开 .采用 CM-纤维素吸附 ,用稀盐酸解吸附 ,得到纯度为92 %的降钙素前体多肽 s CT- Gly.氨基酸的序列分析结构表明 ,重组 s CT- Gly的 N端 1 0个氨基酸与预期一致 .在强酸性条件下 ,没有发生氨基酸的脱酰胺反应 ,氨基酸组成分析与预期基本一致 .质谱法测定的分子量为 3492 ,毛细管电泳测定的等电点 p I为 6.46,大鼠降钙素比活性为 1 90 IU/mg左右 ,与天然的人降钙素相当 .     

Determination of the proteolytic processing sites in the polyprotein encoded by the bottom-component RNA of cowpea mosaic virus

We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high-pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components.     

A new method for affinity chromatography of heme-binding proteins: Synthesis and characterization of hematin- and hematoporphyrin-agarose

Two new affinity columns, using hematin and hematoporphyrin as ligands, have been prepared. Both were made by first attaching 1,6-diaminohexane to Sepharose 4B by the cyanogen bromide procedure and then coupling the porphyrins to the free amino groups of this arm with carbodiimide. This second reaction was done in dimethylformamide to increase the solubility of the porphyrins at pH 4.7. This resin was then washed extensively with dimethylformamide to remove all of the unreacted porphyrin. The new affinity columns are able to bind apoglobin, albumin, and hemopexin, which demonstrates their ability to purify heme-binding proteins. The proteins could be removed by washing the column with a deforming buffer, such as 8 m urea or 0.1 m sodium citrate buffer at pH 4. Neither lysozyme nor hemoglobin bound to these resins demonstrating that the absorbants are specific for apo heme-binding proteins.     

Presence in Leukemic Cells of Avian Myeloblastosis Virus-Specific DNA Sequences Absent in Normal Chicken Cells

(3)H-labeled 35S RNA from purified avian myeloblastosis virus (AMV) was exhaustively hybridized with an excess of normal chicken DNA to remove all viral RNA sequences which are complementary to DNA from uninfected cells. The [(3)H]RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded [(3)H]RNA. The residual RNA hybridized to leukemic chicken DNA but did not rehybridize with normal chicken DNA. This demonstrates conclusively that DNA from AMV-induced leukemic cells contain viral-specific sequences which are absent in DNA from normal cells.     

The binding of an avian myeloblastosis virus basic 12,000 dalton protein to nucleic acids.

The binding of a basic 12,000 dalton protein (p12) from avian myeloblastosis virus to viral RNA and heterologous DNA has been investigated. The binding stoichiometries and constants were determined by an extrinsic fluorescence assay. In both cases each bound p12 molecule occupies four nucleotides and the apparent binding constant is approximately 1 x 10(6) M-1. Binding is non-cooperative and there is no apparent difference in the interaction of p12 with viral RNA or heterologous single-strand DNA. The relative binding constant at various ionic strengths was assayed by the nitrocellulose filter procedure. Analysis of the data revealed that each bound p12 molecule forms three ion pairs with the nucleic acid.     

Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12, at Serine 40, the primary site of phosphorylation in vivo

The major nucleocapsid protein of avian retroviruses, pp 12, binds to single-stranded viral RNA with high affinity. Phosphorylation at Ser-40 is necessary for this binding. In order to examine the role of phosphorylation of serine 40 in the biological function of pp 12, we have introduced a series of amino acid substitutions at this position in the Rous sarcoma virus (Pr-C) protein. Substitution of threonine, alanine, or three other amino acids for Ser-40 had very little or no detectable effect on viral replication, nor did the control substitution of glycine for Ser-43, a nonphosphorylated residue. In vivo and in vitro, the Ala-40 and probably the Thr-40 substituted p 12 proteins are phosphorylated at alternative sites which are phosphorylated to a minor extent in vivo in the wild type protein. A study of the RNA binding properties of Ala-40 substituted p 12 has indicated that the protein has been stabilized in a high affinity RNA binding state which is independent of phosphorylation. The viability of the Ala-40 mutant virus indicates that this high binding affinity may be required for biological activity.