Evidence of methylation of B77 avian sarcoma virus genome RNA subunits.

B77 avian sarcoma virus RNA was labeled with (methyl-3H) methionine under conditions that prevent non-methyl incorporation of 3H radioactivity into purine rings. From the determined values for the extent of methylation of 4S RNA isolated from infected chicken embryo cells, it was estimated that 30 to 40S RNA subunits that results from heat denaturation of the 60 to 70S RNA contain approximately 21 methyl groups, of which 14 to 16 are present at internal positions as N6 -methyladenosine residues. In addition, each of the virion RNA subunits appears to contain about two methyl groups in the "capped" 5' -terminal structure m7G(5')ppp(5') gm. These properties are consistent with the hypothesis that the 30 to 40S genome RNA os oncornaviruses also serves an mRNA function in infected cells.     

Structure of B77 sarcoma virus RNA: stabilization of RNA after packaging.

Extracellular maturation of Bratislava 77(B77) sarcoma virus RNA involves a stabilization of linkage between 35S subunits since the Tm of 60 to 70S RNA in the presence of 0.5 M NaCl increases from 56 to 67.5 C as the age of the virus increases. This stabilization process is strongly temperature dependent; the rate at 45 C is increased fourfold over the rate at 37 C. As a result of the instability of the immature RNA, denaturation to subunits occurs at room temperature during phenol extraction. This dissociation can be prevented by increasing the NaCl concentration during the extraction. The data support a model which proposes that RNA subunits of oncornaviruses exist as assembled 60 to 70S RNA even in immature virions but that the linkages between subunits are stabilized as a function of time. These linkages appear to be maintained by nucleotide base pairing rather than by protein. However, isolated RNA does not undergo stabilization, suggesting that some other component of the virion is necessary for the process to occur.     

Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA.

Cycloleucine, a competitive inhibitor of ATP: L-methionine S-adenosyltransferase in vitro, has been used to reduce intracellular concentrations of S-adenosylmethionine and by this means to inhibit virion RNA methylation in chicken embryo cells that are infected with B77 avian sarcoma virus. Under conditions of cycloleucine treatment, where virus production as measured by incorporation of radioactive precursors or by number of infectious particles is not significantly affected, the internal m6A methylations of the avian sarcoma virus genome RNA are inhibited greater than 90%. The predominant 5'-terminal structure in viral RNA produced by treated cells in m7G(5')pppG (cap zero) rather than m7G-(5')pppGm (cap 1). It appears from these results that internal m6A and penultimate ribose methylations are not required for avian sarcoma RNA synthesis and function. Furthermore, these methylations are apparently not required for transport of genome RNA to virus assembly sites. The insensitivity of the 5'-terminal m7G methylation to inhibition by cycloleucine suggests that the affinity of S-adenosylmethionine for 7-methylguanosine methyltransferase is significantly greater than for the 2'-0-methyltransferases or the N6-methyltransferases.     

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 $\text{p12}\text{15}$, upon injection of avian sarcoma virus 70S or 35S RNA into $\text{Xenopus}$ oocytes has been presented. Further, we show that tRNA^trp primer, bound to 35S RNA, does not block translation of virion RNA under these conditions.     

Sequence relatedness between the subunits of avian myeloblastosis virus reverse transcriptase.

One- and two-diminsional tryptic and chymotryptic peptide maps of 125-I-labeled alpha and alphabeta avian myeloblastosis virus DNA polymerase demonstrate that the alpha polypeptide of the one and two subunit enzymes are structurally similar, if not identical. Furthermore, the beta subunit contains the same major 125I-labeled peptides as alpha, plus several additional peptides. These relationships and the fact that aging of purified alphabeta avian myeloblastosis virus DNA polymerase increases the proportion of alpha DNA polymerase that can be isolated from the alphabeta enzyme by phosphocellulose chromatography, suggests that alpha is derived from beta by proteolytic cleavage.     

Characterization of c-lil, a chicken cellular sequence associated with a stock of B77 avian sarcoma virus.

Using biochemical methods, we have shown that a new specific sequence, v-lil, is associated with a given stock of B77 avian sarcoma virus (clone 9). We prepared a DNA complementary to v-lil sequences, using substractive hybridizations, and investigated the properties of this sequence. v-lil has a genetic complexity of ca. 2,000 nucleotides and is not present in various stocks of avian sarcoma virus, avian leukosis virus, or defective leukemia virus. v-lil is not associated with B77 avian sarcoma virus isolated from the original tumor and thus has been acquired by in vitro passage of the virus on chicken embryo fibroblasts. A search for the origin of the v-lil sequence among the DNAs of different avian species has shown that a similar sequence, c-lil, is present in normal chicken DNA (1 to 2 copies per haploid genome). c-lil is not highly conserved but is present in the DNA of all chickens from the genus Gallus. The c-lil sequence is transcribed at a low level (1 to 3 copies per cell) in normal chicken embryo fibroblasts. The biological function, if any, of v-lil or its cellular equivalent has yet to be determined.     

Sequence homology between Moloney murine sarcoma virus and Moloney leukemia virus RNA.

The Moloney murine sarcoma-leukemia virus [M-MSV (MuLV)], propagated at high multiplicity of infection (MOI), was demonstrated previously to contain a native genome mass of 4 X 10(6) daltons as contrasted to a mass of 7 X 10(6) daltons for Moloney murine leukemia virus (M-MuLV). The 4 X 10(6)-dalton classof RNA from M-MSV (MuLV) was examined for base sequence homology with DNA complementary to the 7 X 10(6)-dalton M-MuLV RNA genome. Approximately 86% of the M-MSV (MuLV) was protected from RNase digestion by hybridization, whereas 95% of M-MuLV was protected under identical conditions. These results indicate that the small RNA class of high-MOI M-MSV (MuLV) contains little (perhaps 10%) genetic information not present in M-MuLV. Virtually all of the 1.8 X 10(6)-dalton subunits of M-MSV (MuLV) RNA contained regions of poly(A) since 94% of the RNA bound to oligo(dT) cellulose in 0.5 M KCl. This suggests that the formation of the 1.8 X 10(6)-dalton subunits occurs before their packaging into virions and does not result from hydrolysis of intact 3.5 X 10(6)-dalton subunits by a virion-associated nuclease.     

Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy.

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10-6 plus or minus 0.25 times 10-6.     

Infection of resistant avian cells by subgroup B Rous sarcoma virus.

Chicken fibroblasts derived from the H & N flock, which have been characterized as resistant to subgroup B avian oncornaviruses in focus assays, can be infected in suspension shortly after trypsinization by subgroup B sarcoma and leukosis viruses. Once cells are plated, resistance to infection reappears rapidly. C/BE cell suspensions obtained by treatment with EDTA instead of trypsin are not as sensitive to infection. Late interference established by preinfection with subgroup B leukosis viruses is not overcome by trypsinization. In addition to C/BE H & N chicken cells, C/ABE RPRL line 7 cells can also be infected by subgroup B viruses shortly after trypsinization; however, none of the cell types can be made sensitive to subgroup E infection. These results are discussed in relation to current information on the genetic control of resistance to avian oncornaviruses.     

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.     

Spontaneous mutations affecting the host range of the B77 strain of avian sarcoma virus involve type-specific changes in the virion envelope antigen.

Previously it was shown that the host-range gene of the Bratislava strain of avain sarcoma virus (B77 virus) spontaneously mutates with a very high rate. The wild-type B77 virus called B77 virus-II, mutates either to virus that efficiently infects duck cells (B77 virus-III) or to virus that does not mutate to the ability to infect duck cells (B77 virus-I) (Zarling and Temin, 1976). No significant differences in either the virion envelope glycoproteins or other major virion proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, pseudotypes of B77 virus-I with proteins of a transformation-defective mutant of B77 virus-III formed foci efficiently in duck cells. An alteration in the envelope protein of B77 virus-I was demonstrated by experiments in which B77 firus-I was fused into duck cells with UV-irradiated Sendai virus and formed foci. Neutralization experiments further demonstrated that B77 virus host-range mutants have altered type-specific envelope antigens. Thus, the spontaneous mutations in the host-range gene of B77 virus involve changes in the type-specific virion envelope antigen.     

Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus.

The structure and membrane interaction of the internal fusion peptide (IFP) fragment of the avian sarcoma and leucosis virus (ASLV) envelope glycoprotein was studied by an array of biophysical methods. The peptide was found to induce lipid mixing of vesicles more strongly than the fusion peptide derived from the N-terminal fusion peptide of influenza virus (HA2-FP). It was observed that the helical structure was enhanced in association with the model membranes, particularly in the N-terminal portion of the peptide. According to the infrared study, the peptide inserted into the membrane in an oblique orientation, but less deeply than the influenza HA2-FP. Analysis of NMR data in sodium dodecyl sulfate micelle suspension revealed that Pro13 of the peptide was located near the micelle-water interface. A type II beta-turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a conformational flexibility of the IFP in analogy to the N-terminal FP such as that of gp41. A loose and multimodal self-assembly was deduced from the rhodamine fluorescence self-quenching experiments for the peptide bound to the membrane bilayer. Oligomerization of the peptide and its variants can also be observed in the electrophoretic experiments, suggesting a property in common with other N-terminal FP of class I fusion proteins.     

Polymorphism of avian sarcoma virus src proteins.

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.     

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.     

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.     

Subunits of oncornavirus high-molecular-weight RNA. I. Stepwise conversion of 60 S AMV (avian myeloblastosis virus) RNA to subunits

The effect of formamide on the dissociation of aggregate structure of high-molecular-weight RNA of avian myeloblastosis virus, an oncornavirus, was studied. It has been found that the pretreatment with increasing formamide concentration leads to the stepwise conversion of 60 – 70 S RNA molecule to 50 – 54 S and 30 – 40 S components; the 50 – 54 S intermediate is then further converted to 30 – 40 S subunits and smaller heterogenous RNAs. It is suggested that the subunits forming the aggregate RNA molecule of oncornaviruses are held together by not equally stable double stranded regions.     

Effect of aphidicolin on avian sarcoma virus replication.

We studied the effect of aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, on viral DNA replication and integration during the first 24 h after infection of quail embryo fibroblasts with avian sarcoma virus. In drug-treated cells, the synthesis of unintegrated linear viral DNA species was not impaired; however, the subsequent accumulation of circular viral DNA species and integrated proviral DNA was reversibly inhibited. After removal of the drug, circular viral DNA species were derived from preexisting linear viral DNA species, instead of being derived by de novo synthesis.