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PubMed1998

Structural basis for inactivating mutations and pH-dependent activity of avian sarcoma virus integrase.

Crystallographic studies of the catalytic core domain of avian sarcoma virus integrase (ASV IN) have provided the most detailed picture so far of the active site of this enzyme, which belongs to an important class of targets for designing drugs against AIDS. Recently, crystals of an inactive D64N mutant were obtained under conditions identical to those used for the native enzyme. Data were collected at different pH values and in the presence of divalent cations. Data were also collected at low pH for the crystals of the native ASV IN core domain. In the structures of native ASV IN at pH 6.0 and below, as well as in all structures of the D64N mutants, the side chain of the active site residue Asx-64 (Asx denotes Asn or Asp) is rotated by approximately 150 degrees around the Calpha---Cbeta bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bonds with the amide group of Asn-160, and thus, the usual metal-binding site, consisting of Asp-64, Asp-121, and Glu-157, is disrupted. Surprisingly, however, a single Zn2+ can still bind to Asp-121 in the mutant, without restoration of the activity of the enzyme. These structures have elucidated an unexpected mechanism of inactivation of the enzyme by lowering the pH or by mutation, in which a protonated side chain of Asx-64 changes its orientation and interaction partner.

Amino Acid Sequence↗
PubMed1984

Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins.

Two monoclonal antibodies have been obtained that recognize antigenic determinants within the C-terminal fps-encoded region of P140gag-fps, the transforming protein of Fujinami avian sarcoma virus (FSV). The hybridomas which secrete these antibodies (termed 88AG and p26C) were isolated after the fusion of NS-1 mouse myeloma cells with B lymphocytes from Fischer rats that had been immunized with FSV-transformed rat-1 cells. FSV P140gag-fps immunoprecipitated by either antibody is active as a tyrosine-specific kinase and is able to autophosphorylate and to phosphorylate enolase in vitro. The fps-encoded proteins of all FSV variants, including the gag- p91fps protein of F36 virus, are recognized by both monoclonal antibodies. However, the product of the avian cellular c-fps gene. NCP98, and the transforming proteins of the recently isolated fps-containing avian sarcoma viruses 16L and UR1 are recognized only by the p26C antibody. The 88AG antibody therefore defines an epitope specific for FSV fps, whereas the epitope for p26C is conserved between cellular and viral fps proteins. The P105gag-fps protein of the PRCII virus is not precipitated by p26C (nor by 88AG), presumably as a consequence of the deletion of N-terminal fps sequences. These data indicate that the fps-encoded peptide sequences of 16L P142gag-fps and UR1 P150gag-fps are more closely related to NCP98 than that of FSV P140gag-fps. This supports the view that 16L and UR1 viruses represent recent retroviral acquisitions of the c-fps oncogene. The P85gag-fes transforming protein of Snyder-Theilen feline sarcoma virus is not precipitated by either monoclonal antibody but is recognized by some antisera from FSV tumor-bearing rats, demonstrating that fps-specific antigenic determinants are conserved in fes-encoded proteins.

Animals↗
PubMed1978

Cell-mediated cytotoxicity against hamster cells transformed by avian sarcoma viruses. 2. Sensitivity of different subclones.

The presence of new antigens on different subclones of hamster cells transformed by avian sarcoma viruses was investigated by an in vitro cellular microcytotoxicity assay. The RS 2/3 and RS 2/10 subclones derived from the RS 2 clone, transformed with a Schmidt-Ruppin strain of RSV were highly sensitive to the cytotoxic action of spleen effector cells obtainled from hamsters immunized with RSV-transformed rat cells. The RS 2/6 subclone was sensitive only when 1,000 target cells were seeded and the RS 2/7 subclone was not susceptible to the cytotoxic action of effector cells. The RB 12/1 subclone, derived from the clone RB 12, transformed with a Bryan strain of RSV was poorly sensitive. The comparison between the sensitivity to XC-sensitized spleen cells of the different subclones and their other known properties was discussed.

Animals↗
PubMed1975

Neoplastic transformation of mouse embryo cells by virus released from tumorigenic mouse cells transformed by avian sarcoma virus B77.

Mouse C3H embryo cells were transformed in vitro by avian sarcoma virus Bratislava 77 (B77) released scantily from a mouse cell line transformed earlier by the same virus. B77 virus transformed C3H embryo cells contained B77 viral genome and were transplantable into syngeneic as well as allogeneic DBA/2J young mice in which autochthonous sarcomas were induced. Tumors in both strains of mice were virogenic. The probable reasons for an increased transformation capacity of B77 virus in mammals are discussed.

Animals↗
PubMed1979

Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation.

The avian sarcoma virus (ASV) protein responsible for cellular transformation in vitro and sarcomagenesis in animals was studied structurally with special reference to the sites of phosphorylation on the polypeptide. The product of the ASV src gene, pp60src, is a phosphoprotein of 60,000 daltons. We found that pp60src contained two major sites of phosphorylation, one involving phosphoserine and the other involving phosphothreonine and possible addtional minor sites of phosphorylation. By using N-formyl[35S]methionyl-tRNAf as a radiolabeled precursor in the cell-free synthesis of the src protein in conjunction with partial proteolysis mapping, we determined that the major phosphoserine residue was located on the amino-terminal two-thirds of the molecule and that the phosphothreonine was located on the carboxy-terminal third. We further determined that the phosphorylation of pp60src in cell extracts involved at least two protein kinases, the one that phosphorylated the major serine site being cyclic AMP dependent and the other, acting on the threonine residue, being a cyclic nucleotide-independnet phosphotransferase. Finally, analysis of the pp60src isolated from cells infected with a temperature-sensitive src gene mutant of ASV revealed that phosphorylation of the major threonine residue was severely reduced when infected cells were grown at the nonpermissive temperature, whereas a phosphorylation pattern characteristic of the wild-type pp60src was observed at the permissive temperature. As pp60src has an associated protein kinase activity, the possible involvement of phosphorylation-dephosphorylation reactions in the functional regulation of ASV transforming protein enzymatic activity is discussed.

Alpharetrovirus↗
PubMed1981

Long-term preservation of transfecting activity of avian sarcoma proviral DNA.

The integrated proviral DNA of avian sarcoma virus (ASV) in host cell chromosomes has been isolated and stored in saline sodium citrate (SSC) solution or in 70% ethanol at 4 degrees C in a refrigerator over 4 years. This DNA was assayed by transfection of chick embryo cells(CEC). The biological activity of cellular transformation by the stored DNA was compared with that of a fresh isolate of the proviral DNA. The efficiency of the transfection by each DNA was almost the same.

Alpharetrovirus↗
PubMed1980

The purified product of the transforming gene of avian sarcoma virus phosphorylates tyrosine.

The product of the avian sarcoma virus transforming gene (src) is a phosphoprotein of 60,000 daltons (pp60src) which is responsible for the oncogenic potential of the virus. Recent findings indicate that this protein possesses an affiliated protein kinase activity. We have determined by hydrodynamic measurements and gel filtration that this kinase activity tracks with a highly asymmetric molecule of 60,000 daltons, strengthening the idea that pp60src alone (as opposed to a complex) possesses the enzymatic activity. To more fully characterize the properties of this kinase activity, we undertook its purification by two independent methods. In each case, a protein related to pp60src was extensively purified from contaminating cellular proteins. The yields from one of the procedures were sufficient to induce high titer monospecific antibodies against pp60src in mice. We have shown that purified pp60src is able to phosphorylate several protein substrates other than IgG. The conclusion that pp60src possesses the responsible enzymatic activity was strengthened by demonstrating that a temperature-sensitive conditional mutation in src affected the thermal stability of the purified protein. It has recently been shown that the protein kinase activity affiliated with pp60src phosphorylates tyrosine residues on IgG. We have examined the target specificity of the purified protein on several substrates other than IgG, and show that in every case, the phosphorylation occurs exclusively at a tyrosine residue; it therefore appears that tyrosine phosphorylatin is not an artifact of phosphorylation in th immunoprecipitate, but instead represents the general substrate specificity of pp60src.

Alpharetrovirus↗
PubMed2001

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus.

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.

Alpharetrovirus↗
PubMed1980

Antibody-mediated polysome precipitation as a method for the size determination of viral mRNA species: viral envelope glycoprotein mRNA of avian sarcoma viruses.

A method was developed that allows the in situ isolation of viral mRNA, i.e. from polysomes of infected cells. This was achieved by precipitating intact polysomes via their nascent virus polypeptides using virus-specific antibodies. As a model, antibodies to the major envelope glycoprotein (gp85) of avian sarcoma viruses were employed to precipitate those polysomes from infected cells which synthesized the corresponding p70 virus glycoprotein precursor. Normal immunoglobulin and polysomes from uninfected chicken embryo fibroblasts served as controls. The radioactivity labelled mRNA from antibody-precipitated polysomes could subsequently be extracted and characterized for size. It was found that avian sarcoma virus gp85 envelope glycoprotein is predominantly synthesized by a 22--28 s viral mRNA. In addition, minor amounts of gp85-specific mRNAs of 16--21 s and 28--35 s could be demonstrated. The data indicate the presence in polysomes of viral mRNA species coding for i) gp85 only (16--21 s RNA), ii) gp85 and pp60src protein from the adjacent src-gene (22--28 s RNA), and iii) a large viral precursor protein (28--35 s RNA).

Animals↗
PubMed1987

The methylation state of the proviruses in avian sarcoma virus transformed chick and rat cells.

The endogenous viruses in the avian cells are not completely methylated, nor are the Schmidt-Ruppin RSV-D (SRD) proviruses in the infected cells completely unmethylated. Avian sarcoma proviruses integrated in rat transformed cloned cells are heavily methylated. In these cells, a region in the 3' end of the env gene is unmethylated in all the src-containing proviruses but not in the transformed defective (td) proviruses. A possible role for the hypomethylation of the 3' end of the env region is proposed.

Animals↗
PubMed1984

Two strains of avian sarcoma virus newly isolated from chick fibrosarcomas induced by lymphatic leukemia virus subgroup A in two lines of chickens.

Two strains of avian sarcoma virus, designated S1 and S2, have been newly isolated from avian lymphatic leukemia virus (LLV) subgroup A [LLV(A)]-induced fibrosarcomas in 2 chickens from 2 White Leghorn flocks of lines 151 and BK. Each stock of S1 and S2 contained 2-3 X 10(3) focus-forming units of virus/ml and 10(6) tissue culture infective dose of LLV(A)/ml. The focus-forming titer of S1 and S2 estimated in chick embryo fibroblast cultures was almost consistent with that estimated by tumor (fibrosarcoma) formation in the chicks. All of the chicks bearing wingweb tumors (fibrosarcomas at the site of inoculation) had macroscopically neoplastic lesions (fibrosarcomas) in a number of visceral organs, such as the lung, liver, and spleen. Each stock of S1 and S2 was cloned by picking a single focus, and each focus was examined for the production of focus-forming virus and LLV. The results suggest that S1 and S2 are heterogenous stocks of sarcoma viruses that are replication-defective to various degrees. The low titer of focus-forming virus in the stocks of S1 and S2 was considered to be due to the small amounts of avian sarcoma virus that could be complemented by LLV.

Animals↗
PubMed1987

Avian sarcoma virus 17 carries the jun oncogene.

Biologically active molecular clones of avian sarcoma virus 17 (ASV 17) contain a replication-defective proviral genome of 3.5 kilobases (kb). The genome retains partial gag and env sequences, which flank a cell-derived putative oncogene of 0.93 kb, termed jun. The jun gene lacks preserved coding domains of tyrosine-specific protein kinases. It also shows no significant nucleic acid homology with other known oncogenes. The probable transformation-specific protein in ASV 17-transformed cells is a 55-kDa gag-jun fusion product.

Amino Acid Sequence↗
PubMed1982

Cell surface alterations of avian sarcoma virus B77-and 3,4-benzo(a)pyrene-transformed rat fibroblasts. II. Cell surface glycoproteins characterized by two-dimensional electrophoresis.

Cell surface proteins of avian sarcoma virus B77-, 3,4-benzo(a)pyrene- and spontaneously transformed rat fibroblasts were tritium-radiolabeled by mild periodate oxidation followed by tritiated sodium borohydride reduction and by galactoseoxidase/NaB3H4 technique. Radiolabeled cell surface sialoglycoproteins were analyzed by electrophoresis under denaturing conditions (SDS-PAGE), or by two-dimensional electrophoresis (isoelectric focusing, SDS-PAGE). Following major glycoproteins were quantitatively decreased on all examined transformed cells: a 220k glycoprotein with pI of approximately 6.2, a 180k glycoprotein with pI of approximately 6.0-6.2, a 110k glycoprotein (pI 6.2). A series of further sialoglycoproteins was quantitatively increased on all examined transformed cells, as follows: a markedly increased 70k glycoprotein (pI approximately 4.8) and a 120k glycoprotein (pI 5.0-5.2) increased more markedly on onco-virus-transformed and spontaneously transformed cells. Further sialogalactoprotein (28k, pI 5.8) was visualized as increased on all examined transformed cells only by galactoseoxidase/NaB3H4 technique. A 70k sialoglycoprotein was immunoprecipitated from transformed cells by the immune serum against Friend murine leukemia virus envelop glycoprotein gp70 and, to a markedly lesser extent, from untransformed cells. A similar glycoprotein, together with an another 120k glycoprotein were precipitated from transformed cells and to a minor extent from untransformed cells also by an antiserum against endogenous rat type-C virus.

Animals↗
PubMed1981

Tandem duplication of the proviral DNA in an avian sarcoma virus-transformed quail clone.

Previous study has shown that an avian sarcoma virus-transformed quail clone, Q-B77-11, apparently contains full-size proviral DNA and releases virus particles which contain polymerase activity, but are unable to form foci. Recently, we have found that after extended tissue culture of Q-B77-11, the predominant cell type changed from one in which there was a single copy of the integrated viral DNA to one with two copies in tandem. There was approximately one copy of the large terminal redundancy between these two tandem proviruses. Polyadenylic acid-containing RNA species of twice the size of the viral genome were detected in these cells and are interpreted as transcripts of the tandem provirus.

Animals↗
PubMed1985

Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25.

The newly isolated avian sarcoma viruses, ASV 9, 17, and 25, cause fibrosarcomas in young chickens and induce foci of transformed cells in chick embryo fibroblast cultures. They are defective in replication and belong to envelope subgroup A. The sizes of their genomes are 6 kb (ASV 9), 5 kb (ASV 17), and 6 kb (ASV 25), respectively. All three contain long terminal repeat (LTR) and gag sequences but lack pol. env is absent from ASV 9 and ASV 25, but some env sequences are detectable in ASV 17. None of the defective viral genomes hybridized to selected onc probes representing src, fps, yes, myc, myb, and erb A. erb B appears absent from ASV 9 and ASV 17, but some hybridization between the erb B probe and the RNA of ASV 25 was detected. ASV 9 codes for a transformation-specific gag-linked protein of 130kDa. Multiple gag-linked transformation-specific proteins are seen in ASV 17 and 25; they require further study.

Animals↗
PubMed1995

Multimerization determinants reside in both the catalytic core and C terminus of avian sarcoma virus integrase.

We have shown previously that the active form of avian sarcoma virus integrase (ASV IN) is a multimer. In this report we investigate IN multimerization properties by a variety of methods that include size exclusion chromatography, chemical cross-linking, and protein overlay assays. We show that removal of the nonconserved C-terminal region of IN results in a reduced capacity for multimerization, whereas deletion of the first 38 amino acids has little effect on the oligomeric state. Binding of a full-length IN fusion protein to various IN fragments indicates that sequences in both the catalytic core (residues 50-207) and a C-terminal region (residues 201-240) contribute to IN self-association. We also observe that the isolated C-terminal fragment (residues 201-286) is capable of self-association. Finally, a single amino acid substitution in the core domain (S85G) produces a severe defect in multimerization. We conclude from these analyses that both the catalytic core and a region in the nonconserved C terminus are involved in ASV integrase multimerization. These results enhance our understanding of intergrase self-association determinants and define a major role of the C-terminal region of ASV integrase in this process.

Avian Sarcoma Viruses↗
PubMed1979

Analysis of cellular integration sites in avian sarcoma virus infected duck embryo cells.

The cellular sites of integration of avian sarcoma virus (ASV) have been examined in clones of duck embryo cells infected with the Bratislava 77 strain of ASV using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled ASV complementary DNA probes. DNA prepared from 11 clones of duck embryo cells infected with the Bratislava 77 strain of ASV was digested with the restriction enzymes HpaI, which cleaves once within the viral genome, and Hind III, which cleaves twice within the viral genome, and the virus-cell DNA juncture fragments were resolved by agarose gel electrophoresis. Analysis of the virus-cell junctures present in individual ASV-infected duck embryo clones revealed that all clones contain at least one copy of nondefective proviral DNA with some clones containing as many as 5 to 6 copies of proviral DNA. A comparison of the virus-cell juncture fragments present in different ASV-infected clones showed that each clone contains a unique set of virus-cell junctures. These data suggest that ASV DNA can integrate at multiple sites within the duck embryo cell genome and that these sites appear to be different as defined by digestion with the restriction enzymes HpaI and HindIII.

Animals↗
PubMed1979

Evidence for the identity of shared 5'-terminal sequences between genome RNA and subgenomic mRNA's of B77 avian sarcoma virus.

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.

Avian Sarcoma Viruses↗