Biochemical properties of p15-associated protease in an avian RNA tumor virus.

It was observed that the viral structural protein p15 from avian myeloblastosis virus emerges from ion-exchange column chromatography along with a proteolytic activity. p15 is apparently pure, as judged by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and isoelectric focusing. Increase and decrease in proteolytic activity coincided exactly with increasing and decreasing amounts of p15 during ion-exchange chromatography and during size fractionation by gell filtration. The proteolytic activity cleaved various substrates such as bovine serum albumin, ovalbumin, concanavalin A, and casein after denaturation by sodium dodecyl sulfate and heat. Highest enzyme activity was observed around pH 5.7. As judged from its cleavage pattern and its response to proteolytic inhibitors, the proteolytic activity appears papain-like, and the protease responsible for it may be classified as a thiol protease. If added to immunoprecipitated viral polyprotein precursor Pr76, p15 resulted in cleavage of Pr76,which could be inhibited by antibodies against p15.     

Fine structure mapping of an avian tumor virus RNA by immunoelectron microscopy.

The RNA of a deleted strain (lacking Src gene) of an avian sarcoma virus (ASV) was examined by a newly developed immunoelectron microscopic procedure which uses anti-nucleotide antibodies as probes. After denaturation of the RNA and reaction with a high affinity, highly specific anti-7-methylguanosine-5'-phosphate (anti-pm 7G), 81% of 106 molecules examined were found to have antibody at one terminus, in agreement with the presence of a pm 7G cap in ASV-RNA. Hapten inhibition by pm 7G could be demonstrated. Experiments with anti-A and with anti-poly A gave results consistent with the known structure of ASV-RNA, in particular the presence of a 3' poly A tail. These studies illustrate the feasibility of using anti-nucleotide antibodies in a combined immunochemical and electron microscopic study of the fine structure of nucleic acids.     

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.     

Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates.

Previous studies by Guntaka et al. have shown that the unintegrated DNA intermediates of avian RNA tumor virus replication can be readily isolated from cultures of the quail tumor line QT-6 at 1 day after infection. The intermediates include double-stranded linear and covalently closed circular DNA species. Using the analysis procedure of Southern together with previously obtained information regarding the sites of action of certain restriction endonucleases on avian sarcoma virus DNA, we have further characterized the viral DNA intermediates. Evidence is presented that, relative to the RNA genome, most of the linear species possess a direct terminal sequence redundancy equivalent to 0.5 X 10(6) +/- 0.3 X 10(6) daltons of double-stranded DNA. Some of the circular forms also possess a sequence redundancy of 0.21 X 10(6) +/- 0.03 X 10(6) daltons.     

Size of murine RNA tumor virus-specific nuclear RNA molecules.

About 1% of the total RNA of cell lines producing murine leukemia virus is virus-specific RNA. About one-third of the virus-specific RNA is located within the nucleus. The size distribution of virus-specific RNA was determined before and after denaturation. Before denaturation, virus-specific RNA sequences sedimented as a heterogeneous population of RNA molecules, some of which sedimented very rapidly. After denaturation, most of the virus-specific RNA had a sedimentation coefficient of 35S or lower, but a small fraction of the nuclear virus-specific RNA sedimented more rapidly than 35S RNA even after denaturation.     

Cleavage of p15 protein in vitro by human immunodeficiency virus type 1 protease is RNA dependent.

The human immunodeficiency virus (HIV) gag polyprotein is processed by the viral protease to yield the structural proteins of the virus. One of these structural proteins, p15, and its protease cleavage products, p7 and p6, are believed to be responsible for the viral RNA binding which is prerequisite for assembly of infectious virions. To better understand potential interactions between viral RNA, p15, and the HIV protease, we have synthesized p15 in an in vitro system and studied its processing by the viral protease. Using this system, we demonstrate that p15 synthesized in vitro is properly cleaved by the HIV protease in an RNA-dependent reaction. Mutation of cysteine residues in either zinc-binding domain of the p7 portion of p15 does not alter the RNA-dependent cleavage, but mutation of three basic residues located between the zinc-binding domains blocks HIV protease susceptibility. The results support a previously unrecognized role for the interaction of RNA and nucleocapsid-containing gag precursors that may have important consequences for virus assembly.     

Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus.

The rate of avian leukosis virus (ALV)-specific RNA synthesis has been examined in bot- uninfected and ALV-infected synchronized chicken embryo fibroblasts. RNA from cells labeled for 2h with [3H]uridine was hybridized with avian myeloblastosis virus poly(dC)-DNA, and the hybridized RNA was analyzed with poly(I)-spephadex chromatography. Approximately 0.5% of the RNA synthesized in ALV-infected cells was detected as virus specific, and no more than a twofold variation in the rate of synthesis was detected at different times in the cell cycle. In synchronized uninfected chicken embryo fibroblasts, approximately 0.03% of the RNA synthesized was detected as virus specific, and no significant variation in the rate of synthesis was observed during the cell cycle. Treatment of ALV-infected chicken embryo fibroblasts with cytosine arabinoside or colchicine was used to block cells at different stages in the cell cycle. The rates of virus-specific RNA synthesis in cells so treated did not differ significantly from the rates in either stationary or unsynchronized virus-infected chicken embryo fibroblasts. These findings support the conclusion that after the initial division of an ALV-infected chicken embryo fibroblast and the initiation of virus RNA synthesis, the rate of virus-specific RNA synthesis is independent of the cell cycle.     

Genome structure of avian sarcoma virus Y73 and unique sequence coding for polyprotein p90.

The genome structure of a newly isolated sarcoma virus, Y73, was studied. Y73 is a defective, potent sarcomagenic virus and contains 4.8-kilobase (kb) RNA as its genome; in contrast, helper virus associated with Y73 had 8.5-kb RNA, similar to other avian leukemia viruses. Fingerprinting analysis these RNAs demonstrated that the 4.8-kb RNA contains a specific RNA sequence of 2.5 kb, which represents the transforming gene (yas) of Y73. This specific sequence was mapped in the middle of the genome and had at both ends 1- to 1.5-kb sequences in common with Y73-associated virus RNA. This structure is very similar to those of avian and mammalian leukemia viruses. In vitro translation of the 4.8-kb RNA and the immunospecificity of the products directly demonstrated that polyprotein p90, containing p19, is a product translated from capped 4.8-kb RNA and that the specific peptide portion is coded by the yas sequence. Protein 90, which was also found in cells transformed with Y73, was suggested to be a transforming protein.     

Purification of virus-specific RNA from chicken cells infected with avian sarcoma virus: identification of genome-length and subgenome-leghth viral RNAs.

Avian sarcoma virus (ASV)-specific RNA was purified from ASV-infected cells by using hybridization techniques which employ polydeoxycytidylic acid-elongated DNA complementary to ASV RNA as well as chromatography on polyinosinic acid-Sephadex columns. The purity and nucleotide sequence composition of purified, virus-specific RNA were established by rehybridization experiments and analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. Polyadenylic acid-containing RNA purified from ASV-infected cells contained approximately 1 to 4% virus-specific RNA, compared with 0.06 to 0.15% observed in uninfected cells. Sucrose gradient analysis of virus-specific RNA isolated from ASV-infected cells revealed two major classes of polyadenylated viral RNA with sedimentation values of 36S and 26-28S. Cells infected with transformation-defective ASV (virus containing a deletion of the sarcoma gene) contained 34S and 20-22S viral RNA species. Double-label experiments employing infected cells labeled initially for 48 h with [3H]uridine and then for either 30, 60, or 240 min with [32P]phosphate showed that the intracellular accumulation of genome-length RNA (36S) was significantly faster than that of the 26-28S viral RNA species.     

Transfer of defective avian tumor virus genomes by a Rous sarcoma virus RNA packaging mutant.

SE21Q1b, a Rous sarcoma virus mutant which packages cellular rather than viral RNA, is competent for infection of quail cells and can transmit defective transforming retrovirus genes. Stably transformed recipient clones have been obtained by using this mutant.     

Specific site of action for single-strand specific nuclease on the double-stranded circular DNA intermediates of an avian RNA tumor virus.

Circular viral DNA intermediates obtained from the quail tumor line, QT6, at 1 day after infection, were opened at one specific location by the single-strand specific nuclease, S1, of Aspergillus oryzae. This site was no longer accessible to the S1 nuclease when circles were first opened at another location with a restriction endonuclease.     

Analysis of precursors to the envelope glycoproteins of avian RNA tumor viruses in chicken and quail cells.

Immune precipitation with monospecific antiserum was employed to study the intracellular synthesis of viral glycoproteins gp85 and gp37. Labeled gp85 and gp37 were detected from lysates of cells transformed with Rous sacroma virus, strain B77, after long-term labeling with radioactive glucosamine or phenylalanine. Immune precipitates prepared from lysates of cells pulse-labeled for a short time resulted in a glycoprotein of 92,000 molecular weight (gp92). This precursor was stable in B77-transformed Japanese quail cells for several hours, whereas in chicken cells it could be chased within a few hours into virion glycoproteins gp85 and gp37. Similarly, the precursor for the structural viral proteins, pr76, persisted in quail cells much longer than in chicken cells. During very short pulses or in the presence of a glucosamine block (25 mM glucosamine), the antiserum against the viral envelope glycoproteins detected a precursor of higher electrophoretic mobility of approximately 70,000 molecular weight, "p70." Fucose label entered gp92 and gp85 as well as "p70." Proteolytic treatment of virion-bound gp85 in vitro generated two discrete glycoproteins of 62,000 and 45,000 molecular weight, but did not result in an increase in the amount of gp37.     

Processing of avian retroviral gag polyprotein precursors is blocked by a mutation at the NC-PR cleavage site.

The avian sarcoma and leukosis viruses (ASLV) encode a protease (PR) at the C terminus of gag which in vivo catalyzes the processing of both gag and gag-pol precursors. The studies reported here were undertaken to determine whether PR is able to cleave these polyproteins while it is still part of the gag precursor or whether the release of its N terminus to form free PR is necessary for full proteolytic activity. To address this question, we created a mutation that disrupts the PR cleavage site between the NC and PR coding regions of the gag gene. This mutation was introduced into a eukaryotic vector that expresses only the gag precursor and into an otherwise infectious clone of ASLV that carries the neo gene as a selectable marker. These constructs were expressed in monkey COS cells or in quail QT35 cells, respectively. Processing was impaired in both systems. Mutant particles were formed, but they contained no mature processed gag proteins. We observed only the uncleaved gag precursor polypeptide Pr76 in one case or Pr76 and a cleaved product of about 60 kDa in the other. Processing of the mutant gag precursor could be complemented in trans by from a wild-type construct, suggesting that the mutation did not induce gross structural alterations in its precursor. Our results suggest that the PR first must be released from its precursor before it can attack other sites in the gag and gag-pol polyproteins and that cleavage at the NC-PR boundary is a prerequisite for the initiation of the PR-directed processing.     

Influence of defective virion core proteins on RNA maturation with an avian sarcoma virus.

The RNA of the avian sarcoma virus B77 temperature-sensitive mutant LA334 was investigated using electrophoretic analysis. The RNA from mutant virus grown at the nonpermissive temperature (42degrees C) showed a heterogeneous peak between 80 and 125S, and another at about 35S. The RNA of the mutant virus grown at the permissive temperature (35 degrees C) behaved like wild-type B77 virus RNA, exhibiting a major peak at 70S. The homology between the various RNA fractions and virus-specific DNA probe was determined, indicating that mutant virus grown at the nonpermissive temperature contains relatively large amounts of nonviral-specific RNA.