Avian Sarcoma Virus RNA Research Articles

A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.

Two distant upstream regions containing cis-acting signals regulating splicing facilitate 3'-end processing of avian sarcoma virus RNA.

Retroviruses, pararetroviruses, and related retrotransposons generate terminally redundant RNAs by transcription of a template flanked by long terminal repeats in which initiation occurs within the 5' long terminal repeat sequences and 3'-end processing occurs within the 3' long terminal repeat sequences. Processing of avian sarcoma virus RNA is relatively inefficient; approximately 15% of the viral RNA transcripts are read-through products; i.e., they are not processed at the viral poly(A) addition site but at sites in the cellular sequence further downstream. In this report, we show that the efficiency of processing at the viral site is further reduced by deletion of two distant upstream sequences: (i) a 606-nucleotide sequence in the gag gene containing a cis-acting negative regulator of splicing and (ii) a 136-nucleotide sequence spanning the env 3' splice site. The deletion of either or both upstream regions increases the levels of read-through products of both unspliced and spliced viral RNA. In contrast, deletion of the src 3' splice site does not affect the efficiency of processing at the viral poly(A) addition site. The effects on 3'-end processing are not correlated either with distance from the promoter to the poly(A) addition site or with the overall level of viral RNA splicing. Substitution of the avian sarcoma virus poly(A) signal with the simian virus 40 early or late poly(A) signal relieves the requirement for the distant upstream sequences. We propose that cellular factors, which may correspond to splicing factors, bound to the upstream viral sequences may interact with factors bound at the avian sarcoma virus poly(A) signal to stabilize the polyadenylation-cleavage complex and allow for more efficient 3'-end processing.

Interaction between retroviral U5 RNA and the T psi C loop of the tRNA(Trp) primer is required for efficient initiation of reverse transcription.

The 5' end of avian sarcoma and leukosis virus RNA near the primer binding site forms two RNA secondary structures, U5-inverted repeat (U5-IR) and U5-leader stems, which are required for efficient initiation of reverse transcription. Lying between these two secondary structures is a 7-base sequence that can anneal to the T psi C loop of the tRNA(Trp) primer. Base substitutions in U5 RNA which disrupt this potential interaction result in a defect in the initiation of reverse transcription both in vivo and in vitro. The defect can be complemented in vitro by base substitutions in the primer. The U5 RNA-T psi C interaction is also dependent upon the presence of both the U5-IR and the U5-leader structures. These RNA secondary structures and primer interactions are conserved in other type C and D retroviruses, suggesting that there is a common mechanism for the initiation of reverse transcription in all of these retroviruses.

RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses

We have reinvestigated the ability of the matrix protein (MA) (p19gag) of avian sarcoma and leukemia viruses to interact with RNA. Previous reports claimed on the one hand that MA can bind tightly and with a high degree of specificity to avian sarcoma and leukemia virus RNA in vitro and on the other that it cannot bind to RNA at all. We found that MA purified by any of several methods does bind to RNA, as measured by its ability to cause retention of radioactive RNA on nitrocellulose membranes in a filtration assay. However, this interaction is weak and lacks specificity. The interaction of MA with RNA was barely detectable by classical sedimentation analysis, and from this observation we estimate that the intrinsic MA-RNA association constant is ca. 10(3) M-1, at least 3 orders of magnitude smaller than the constant describing the interaction of the viral nucleocapsid protein (NC) (p12gag) with RNA, ca. 10(6) M-1. Separately purified phosphorylated and nonphosphorylated MA species bound RNA equally. We also found that MA can bind to DNA with an affinity similar to that for RNA. The large quantitative discrepancy between our results and earlier published reports can be traced in part to methods of data analysis.

A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging.

The deletion of a conserved sequence of ca. 30 nucleotides in the 5' noncoding leader region of an avian sarcoma virus DNA clone resulted in a loss of infectivity after transfection of chicken embryo fibroblasts. Genetic and biochemical analysis of a representative mutant demonstrated that the env gene was expressed normally. Thus, viral RNA transcription, splicing, and translation were not impaired. The amount of mutant viral RNA encapsidated into virions, however, was severely reduced despite the presence of helper-virus. We conclude that the deleted sequence is an essential cis-acting packaging signal.

Accumulation of spliced avian retrovirus mRNA is inhibited in S-adenosylmethionine-depleted chicken embryo fibroblasts.

The synthesis and processing of B77 avian sarcoma virus RNA in infected chicken embryo fibroblasts was followed in the presence and absence of cycloleucine, a competitive inhibitor of the synthesis of S-adenosylmethionine and thus an inhibitor of RNA methylations. An increase in the steady-state levels of genome-length RNA and a decrease in the steady-state levels of subgenomic RNA molecules were obtained in the S-adenosylmethionine-depleted avian sarcoma virus-infected cells after 24 h of treatment with the inhibitor. The total number of virus-specific RNA molecules per cell, however, remained relatively constant under either condition. The production of newly synthesized virus-specific RNA in cycloleucine-treated and untreated cells infected with a transformation-defective strain of B77 avian sarcoma virus was followed as a function of [(3)H]uridine labeling time. The accumulation of radioactive genome-length 8.4-kilobase (kb) RNA continued in cycloleucine-treated cells, and virus particle production proceeded at normal rates as previously shown by incorporation of labeled nucleoside precursors or amino acids. In contrast, newly synthesized 3.5-kb subgenomic mRNA, the putative mRNA for the envelope protein precursor, failed to accumulate in the treated cells. The extent of the inhibition in the appearance of the radioactive 3.5-kb RNA was correlated with the extent of the inhibition of viral genomic and cellular mRNA methylations and was a function of the cycloleucine concentration. Under conditions in which the accumulation of 3.5-kb envelope protein mRNA was blocked by the cycloleucine treatment, there were significant increases in the rate of synthesis of the polypeptide products of the genome-length RNA, the precursors to the non-glycosylated gag proteins (Pr76(gag)), and the reverse transcriptase (Pr 180(gag pol)) relative to the rate of synthesis of the envelope protein precursor (gPr 92(env)). These results suggest that there is an S-adenosylmethionine requirement for the splicing, but not for the synthesis, packaging, or messenger function, of avian retrovirus genome-length RNA. Possible reasons for this requirement are discussed.

The terminal redundancy of the retrovirus genome facilitates chain elongation by reverse transcriptase.

Transcription of DNA from the RNA genome of retroviruses by reverse transcriptase involves an unusual translocation of the growing chain from the 5' end to the 3' end of the RNA template. In order to elucidate the mechanism by which this translocation occurs, we have used chain termination to analyze nascent viral DNA synthesized in vitro by avian sarcoma virus, and we have determined the nucleotide sequence of appropriate regions of viral DNA isolated from infected cells and cloned into prokaryotic vectors. Our results provide direct experimental evidence for a previously proposed model in which a short terminal redundancy in viral RNA, and a DNA copy of the redundant sequence, are used to allow the growing DNA chain to move from the 5' to the 3' end of the template. Transcription of avian sarcoma virus RNA with purified reverse transcriptase also generates an anomalous product, a hairpin DNA that arises when the initial DNA transcript folds back on itself to continue synthesis. The foldback is mediated by an inverted repeat of 5 nucleotides in the sequence of nascent DNA. Anomalous hairpin DNA is not produced by detergent-activated virions. Thus, constituents of the virions or the configuration of encapsidated viral RNA must facilitate correct transcription.

Restriction endonuclease mapping of the DNA of Rous-Associated virus 0 reveals extensive homology in structure and sequence with avian sarcoma virus DNA

We have prepared a physical map of the DNA of Rous-associated virus O (RAV-O), an endogenous virus released by certain chicken lines, in order to examine the relationship between the genome of this virus and closely related proviruses endogenous to chickens. Nineteen recognition sties for 11 restriction endonucleases have been mapped on the unintegrated linear and circular forms of RAV-O DNA isolated from acutely infected quail cells. PvuI is the only enzyme tested which does not cleave RAV-O DNA. Most of the sites (18 of 19) occur at similar or identical positions in the DNA of avian sarcoma virus (ASV). Significant differences between the maps of corrseponding regions of RAV-O and ASV DNA are observed only with those endonucleases which recognize sites encoded near the 3′ terminus of ASV RNA ( PvuI and EcoRI). Both ends of RAV-O linear DNA contain sequences copied from both the 3′ and the 5′ ends of viral RNA. Two species of closed circular DNA were found: one the same size as the terminally redundant linear DNA (5.0 × 10 6 M r and the other lacking ca. 0.35 kb from an end of linear DNA. Thus the unintegrated forms of RAV-O DNA appear structurally similar to those of ASV DNA[ Shank, P. R., et al. (1978b) . Cell 15, 1383–1395; Hsu, W., et al. (1978) . J. Virol. 28, 810–818]; presumably one copy of a ca. 0.35-kb terminal repeat unit is lost during formation of the smaller circle from linear DNA. The following paper illustrates the utility of the physical map for differentiating between endogenous proviruses which might or might not have sequence identity with the RAV-O genome.

Reverse transcription of avian sarcoma virus RNA into DNA might involve copying of the tRNA primer.

Some of the double-stranded DNA products from the endogenous reaction of detergent-disrupted virions contain up to 18 3'-terminal nucleotides at a location consistent with their being transcribed from the tRNA primer.

Effect of viral RNase H on the avian sarcoma viral genome during early transcription in vitro

We investigated the influence of viral RNase H on the transcription of the avian sarcoma virus RNA in a virion-associated reaction. The ability of RNase H to degrade the RNA moiety of the initially formed RNA-DNA hybrid at the 5' end of the viral genome was found to be greatly dependent on the exact concentration of nonionic detergent used to activate the reaction. At a detergent concentration optimal for extensive and faithful in vitro transcription of avian sarcoma virus RNA by the virion-associated RNA-dependent DNA polymerase, most of the 5' terminus of the RNA was digested in 30 min at 41 degrees C. At higher than optimal detergent concentrations, however, little of that RNA was digested. We conclude that removal of the 5'-terminal redundancy in the RNA after its transcription into DNA is a prerequisite for base pairing of the DNA to the 3'-terminal redundant sequence. Lack of removal of this sequence leads to incorrect elongation and substantial reduction of DNA synthesis. When tested with a synthetic RNA-DNA hybrid, virion-associated RNase H did not reveal a detergent dependence.

Elongation of DNA complementary to the 5' end of the avian sarcoma virus genome by the virion-associated RNA-dependent DNA polymerase.

RNA-dependent DNA synthesis in a virion-associated reaction has been described as being dependent upon the detergent concentration used for disruption of the virion. In this study, the Triton X-100 concentration was found to affect the elongation of the initially synthesized DNA complementary to the last approximately 100 nucleotides at the 5' end of the RNA (cDNA100). Whereas elongation of cDNA100 increased with time of incubation at the optimal detergent concentration, this process was retarded at higher detergent concentrations. At the optimal detergent concentration, elongated DNA was of low chemical complexity, indicating that extension of cDNA100 occurred at a unique site on the RNA. Higher than optimal detergent concentrations resulted in nonspecific elongation and in DNA of high chemical complexity. This was shown by oligopyrimidine tract analysis. Furthermore, actinomycin D was observed to inhibit the elongation of cDNA100 at the optimal detergent concentration. The nature of the elongation process was elucidated by analysis of DNA synthesized in a virion-associated reaction in the presence of bacteriophage Qbeta RNA. At the optimal detergent concentration DNA complementary only to avian sarcoma virus RNA was synthesized, whereas at higher concentrations DNA was copied from both avian sarcoma virus and Qbeta RNA. We conclude that the elongation mechanism of cDNA100 is affected by the detergent concentration and elongation is unspecific at higher than optimal detergent concentrations. The mechanism by which the nonionic detergent stimulates DNA synthesis has not yet been resolve. We assume that other factors in addition to DNA polymerase are involved in elongation of cDNA100.

Translation of avian sarcoma virus RNA in Xenopus laevis oocytes

Evidence for the synthesis and processing of Pr76 (precursor to group-specific antigens p27, p19, and p1215, upon injection of avian sarcoma virus 70S or 35S RNA into Xenopus oocytes has been presented. Further, we show that tRNAtrp primer, bound to 35S RNA, does not block translation of virion RNA under these conditions.

Cell-free translation of avian sarcoma virus RNA: Suppression of the gag termination codon does not augment synthesis of the joint gag/pol Product

The RNA genome of avian sarcoma virus (ASV) and 38S polyadenylated RNA from cells producing ASV directed the cell-free synthesis of two viral gene products: Pr76 gag, the precursor to structural proteins of the virion core, and P180 gag/pol, a joint product of two viral genes that is the putative precursor of viral reverse transcriptase. To test the hypothesis that P180 gag/pol is synthesized by read-through of the termination codon for gag, we determined the effect of purified yeast suppressor tRNAs on cell-free translation from gag and pol. Although the synthesis of P180 gag/pol was not enhanced by the suppressor tRNAs, the use of amber suppressor tRNA reduced the synthesis of Pr76 gag and resulted in the synthesis of a new 80, 000 dalton polypeptide (P80) that contained the antigenic determinants of gag. Studies using chemical inhibitors of initiation of translation demonstrated that P80 was generated by the addition of amino acids to the carboxy terminus of Pr76 gag. We conclude that the gag gene of ASV is terminated by an amber codon (UAG), and that a second termination signal occurs ∼120 nucleotides downstream. Our data indicate that suppression of a single termination codon does not suffice for the production of P180 gag/pol. Thus it is doubtful that suppression by tRNA is responsible for the synthesis of this protein. The mechanism by which P180 gag/pol is synthesized in infected cells remains uncertain.

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.

An electron microscopic method for the mapping of proteins attached to nucleic acids

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.

Translation of 35S and of subgenomic regions of avian sarcoma virus RNA.

Rabbit antiserum monospecific for an internal structural protein, p27, of avian sarcoma viruses (ASV) was found to immunoprecipitate polypeptides with molecular weights (Mr) of 180,000 and 76,000 from cell-free reticulocyte lysates programmed by ASV 35S RNA and also from lysates of ASV-infected cells. In addition, the Mr 180,000 protein was also precipitated by antiserum raised against virion DNA polymerase, suggesting that is a product of the two genes nearest the 5' end of virion 35S RNA. We have also investigated the ability of subgenomic portions of virion RNA to program cell-free protein synthesis. A 10-12S poly(A)-containing fragment of RNA from both nondefective and transformation-defective ASV directed the synthesis of a polypeptide of Mr 29,000 immunologically unrelated to the gs antigens; 20-24S poly(A)-containing RNA from nondefective ASV directed the synthesis of a polypeptide of Mr 60,000 not found when a similar RNA preparation from transformation-defective ASV was translated, suggesting that it is the product of the ASV src gene. These results indicate that internal initiation sites for protein synthesis exist on the 35S RNA genome.

Terminally repeated sequences in the avian sarcoma virus RNA genome.

The initiation of DNA synthesis in vitro by RNA-directed DNA polymerase (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) of avian oncornaviruses requires a tRNAtrp primer molecule located close to the 5' end of the viral RNA genome. DNA transcripts, 100 nucleotides in length, initiated on the tRNAtrp primer molecule contain nucleotide sequences complementary to a large (25 nucleotides) RNase T1 oligonucleotide, T-13, located at the 5' terminus of the avian sarcoma virus RNA genome. tRNAtrp-initiated DNA transcripts with a length of about 70 nucleotides contain substantially fewer nucleotide sequences complementary to this 5'-terminal oligonucleotide, suggesting that the tRNAtrp primer associated with the avian sarcoma virus RNA is located approximately 100 nucleotides from the 5' end of the RNA. In addition, we present evidence to demonstrate that DNA transcribed from avian sarcoma virus RNA sequences located at the 3' end, immediately adjacent to the poly(A), contains nucleotide sequences that are complementary to the 5'-terminal T1 oligonucleotide T-13. These data indicate that the 5' end of the viral genome contains nucleotide sequences that are repeated at the 3' end of the genome. We conclude that the avian oncornavirus RNA genome is terminally redundant.

Characterization of dna complementary to nucleotide sequences adjacent to poly(A) at the 3′-terminus of the avian sarcoma virus genome

We have prepared DNA complementary to a sequence of 300–400 nucleotides adjacent to the poly(A) at the 3′-terminus of the genome of avian sarcoma virus (ASV) DNA was transcribed from denatured ASV RNA following initiation on the primer (dT) 12–18. We demonstrated that homopolymer synthesis was not a significant feature of our enzymatic reaction, and we proved that most if not all the initiations on (dT) 12–18 occurred at or near the 3'-terminus of the viral RNA. The presence of oligo(dT) on cDNA 3, accelerates hybridization between cDNA 3, and polyadenylated viral RNA; this artifact can be eliminated by including a competing homopolymer [poly(dT) or poly(A)l in the reaction mixtures. Nucleotide sequences complementary to cDNA 3, occur only once or twice in the genome of avian sarcoma viruses, are present in the genomes of virus strains from subgroups A, B, C, D, E, and F of avian leukosis-sarcoma viruses, and are conserved even when large deletions affect an adjacent viral gene ( src) By contrast, we find little or no complementarity between cDNA 3, and the genomes of golden pheasant virus (subgroup G of avian leukosis-sarcoma viruses), mouse mammary tumor virus, and the Moloney strains of murine sarcoma virus and murine leukemia virus. cDNA 3, can be used to identify the 3′-terminus of the avian sarcoma virus genome by molecular hybridization and will therefore be a useful reagent for the analysis of viral replication.

Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits

Following ribonuclease digestion of methyl-3H-labeled B77 avian sarcoma virus RNA subunits, methylated oligonucleotides were isolated by diethylaminoethylcellulose chromotogrpahy. Partial nucleotide sequences were deduced from the known enzymatic specificities of the ribonucleases. In addition to methylated nucleosides in the 5'-terminal cap structure, m7G(5')GmpCp, N6-methyladenosine(m6A) was found to be present in only two internal sequences of the RNA molecule, Gpm6ApC and Apm6ApC. The average numbers of methylated nucleosides per RNA subunit are about 12-13 in Gpm6ApC, 1-2 in Apm6ApC, and 2 in m7GpppGmpCp. The sequences containing m6A in B77 sarcoma virus RNA are identical to m6A-containing sequences previously reported for the bulk mRNA from HeLa cells (Wei, C.M., Gershowitz, A., and Moss, B. (1976), Biochemistry 15, 397-401). Analysis of the oligonucleotides produced by RNase A digestion indicated that the sequence of bases on the 5' side of these trinucleotides is not specific. The oligonucleotide profile, however, was highly reproducible in different virus preparations. This suggests that the methylations occur at specific positions on the RNA molecule. Some of the methylated oligonucleotides produced by RNase A digestion appear to be present in less than molar amounts. Several hypotheses are proposed to explain this result.

Characteristics of virus-specific RNA in avian sarcoma virus-transformed BHK-21 cells and revertants

Several characteristics of viral RNA transcripts in a clone of avian sarcoma virus (ASV)-transformed baby hamster kidney cells have been studied and compared with the characteristics of viral RNA in clones of cells reverted to a normal phenotype. As reported previously [Deng, C. T., Boettiger, D., Macpherson, I., and Varmus, H. E. (1974). Virology 62, 512–5211, both transformed and reverted cells contain one DNA copy of the viral genome per cell, but the concentration of viral RNA is 2- to 85-fold higher in the transformed cells than in the reverted cells. We now show that ASV RNA from cells of both types contains poly(A) sequences, is threefold more abundant in the cytoplasm than in the nuclei, and is associated with polyribosomes. In the transformed clone, whole cell RNA contains 35 and 24 S species of ASV RNA, whereas only 24 S ASV RNA is detected in the cytoplasm; in the reverted clones, the 24 S species of ASV RNA is present in whole cell RNA and in poly(A)-containing cytoplasmic RNA, but the amounts of viral RNA in these cells are too low to determine unambiguously whether they also contain 35 S ASV RNA. Nucleotide sequences specific for the transforming gene of ASV are present in the RNA, including the polyribosome-associated RNA, of both transformed and reverted cell types, but the concentration of these sequences is reduced in the reverted cell and its polyribosomes in proportion to the overall reduction in viral RNA in reverted cells. Thus, a reduction in the concentration of viral RNA may be the only change in viral gene expression in the reverted cells.