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C M Stoltzfus

Publications and source records attributed to C M Stoltzfus.

53 records · Page 3Linked to original sources
PubMed1983

Stabilities of avian sarcoma virus RNAs: comparison of subgenomic and genomic species with cellular mRNAs.

The stabilities of B77 avian sarcoma virus intracellular RNAs were compared to the stability of the total cellular poly(A)-containing RNA by labelling infected chicken embryo fibroblasts with [3H]uridine for 15 h, adding actinomycin D (1 microgram per ml) to block further transcription of viral RNA, and selecting virus-specific RNA from the total cellular poly(A)-containing RNA at 3 hourly intervals. The three virus-specific RNA species (9.3, 3.3 and 5.4 kilobases) decayed with half-lives of 7.5, 10, and 15 h, respectively, whereas the bulk of the cellular mRNA decayed with a half-life of 13 h. To correlate these decay rates with the disappearance of mRNA activities, the actinomycin D-treated cells were pulse-labelled with [3H]leucine at 3 hourly intervals after the addition of the drug and virus-specific protein synthesis was assayed by immunoprecipitation. The mRNA activity for the precursor to the non-glycosylated viral structural proteins (Pr76gag) decayed with a half-life of approximately 6 h, whereas the mRNA activity coding for the precursor to the envelope proteins (gPr92env) decayed with a half-life of 14 h. Thus, the rate of decay of the individual mRNA species corresponded reasonably well with the decay rate for the synthesis of two of the corresponding gene products. The results indicated that the 5.4 kb env mRNA is more stable under these conditions than the 9.3 kb gag mRNA but was not significantly more stable than the bulk of the cellular mRNA. Virus particle production following the addition of actinomycin D was determined by the reverse transcriptase assay and by the incorporation of viral genomic 70S RNA into extracellular virions. Both assays yielded similar results and indicated that particle production was inhibited at a rate (t 1/2 = 4 h) somewhat faster than the decay of Pr76gag synthesis or the disappearance of 9.3 kb RNA. It was established by two independent methods (pulse and chase, and approach to isotope equilibrium), however, that the intracellular half-life of the RNA that is packaged into virions is 6 to 7 h. Thus, these results suggest that a single metabolic pool of 9.3 kb RNA exists in avian sarcoma virus-infected cells and is used both as mRNA and as genome RNA.

Animals↗
PubMed1983

Particles of reduced infectivity and deficient in envelope glycoproteins are produced in cycloleucine-treated B77 avian sarcoma virus-infected chicken embryo fibroblasts.

We have previously shown that the inhibition of methylation reactions by the treatment of B77 avian sarcoma virus-infected cells with medium containing cycloleucine results in an inhibition in the intracellular accumulation of the spliced subgenomic mRNA for the virion envelope protein precursor, whereas the genome-size RNA accumulates in larger than normal amounts (C. M. Stoltzfus and R. W. Dane, J. Virol. 42:918-931, 1982). To measure the production of virus particles, we have now determined the reverse transcriptase activity in the culture fluid from infected cells treated with various concentrations of cycloleucine. The activity was somewhat greater in the fluid from the cycloleucine-treated cells than it was in the fluid from the control cells, suggesting an enhancement of particle production in the presence of cycloleucine. In contrast, the production of infectious virions, as determined by the focus assay, decreased when the cycloleucine concentration of the medium increased. We determined the polypeptide compositions of purified particles produced from infected cells treated with or without cycloleucine and labeled with [(3)H]leucine. The relative amounts of radioactivity associated with p19 and p27 were approximately the same in all of the preparations. In contrast, significant decreases were observed in the relative amounts of [(3)H]leucine radioactivity associated with the virion glycoproteins gp85 and gp37. The extent of the decrease in the ratio of gp85 to p27 was a function of the cycloleucine concentration and correlated well with the decrease in the infectivity of the virus particles. Therefore, it is probable that the observed reduction of specific infectivity results from the reduced amounts of envelope glycoproteins in the particles budding from cycloleucine-treated cells.

Amino Acids↗
PubMed1982

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.

Animals↗
PubMed1981

Selective inhibition of avian sarcoma virus protein synthesis in 3-deazaadenosine-treated infected chicken embryo fibroblasts.

The production of B77 avian sarcoma virions was inhibited more than 90% in infected chicken embryo fibroblasts that were treated with 100 microM 3-deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase and, for this reason, an inhibitor of methylation reactions. This nucleoside analog at a concentration of 100 microM inhibited the rates of overall cellular protein synthesis and polyadenylated RNA synthesis by 40 to 50%. Rates of viral protein synthesis were compared, and the results indicated that in infected cells treated with 3-deazaadenosine syntheses of both the precursor of the gag proteins (pr76gag) and the precursor of the reverse transcriptase (pr180gag pol) were inhibited. Synthesis of the precursor of the viral envelope glycoproteins (pr92env) appeared to be affected less by the analog treatment. Most of the host polypeptides also continued to be synthesized in 3-deazaadenosine-treated cells. The fraction of the total RNA represented by virus-specific RNA in the 3-deazaadenosine-treated cells was approximately 40% of the fraction of the total RNA represented by viral RNA in control cells, as determined by hybridization kinetics. Therefore, there was a selective inhibition of viral RNA accumulation in the presence of 3-deazaadenosine. The amounts of genome-sized 35S and 38S RNAs were reduced compared with the amounts of 28S and 21S viral mRNA's. These results suggest that selective inhibition of the synthesis of viral proteins is due to selective decreases in the amounts of the mRNA's for these polypeptides.

Animals↗
PubMed1979

Processing and function of undermethylated chicken embryo fibroblast mRNA.

Cycloleucine (1-aminocyclopentane-1-carboxylic acid) is a potent inhibitor of RNA methylation in B77 sarcoma virus-infected chicken embryo fibroblasts. Under conditions where 40 mM cycloleucine is present, internal N-6-methyladenosine and 5'-terminal cap 2'-O-ribose methylations of poly(A)+ RNA are inhibited greater than 90%. The methylation of the 5'-terminal 7-methylguanosine, however, does not appear to be significantly affected. The poly(A)+ RNA synthesized in cycloleucine-treated cells is transported from the nucleus to the cytoplasm and associates with polyribosomes at rates comparable to poly(A)+ RNA in untreated cells. On the other hand, the transport and utilization of newly synthesized ribosomal RNA in cycloleucine-treated cells is impaired, and the accumulation of mature 18 S and 28 S rRNA is reduced.

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↗
PubMed1977

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.

Avian Sarcoma Viruses↗
PubMed1976

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.

Adenosine↗
PubMed1976

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.

Base Sequence↗
PubMed1975

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.

Avian Sarcoma Viruses↗
PubMed1974

Origin of reovirus oligo(A).

Reovirus contains about 1,200 molecules per virion of oligo(A) of chain length 10 to 15 nucleotides in addition to the 10 double-stranded genome segments. Virions purified from infected BHK, HeLa, or L cells had similar amounts of oligo(A) of the same composition, indicating that it is a virus-specific product. Although conversion of virions to cores by chymotryptic digestion resulted in an almost complete loss of oligo(A) and a marked decrease in infectivity, the infectivity could be partially restored by adsorbing cores to cells in the presence of Kaopectate. Core-infected cells yielded virions that contained a normal complement of oligo(A). The results indicate that oligo(A) is not essential for infectivity or required as a primer/template for its own synthesis.

Adenosine↗
PubMed1974

Poly(A) polymerase activity in reovirus.

An enzymatic activity which synthesized oligo(A) in vitro was found in highly purified reovirus. The poly(A) polymerase activity was dependent on Mn(2+) and utilized only ATP, whereas the virion-associated RNA polymerase required all four ribonucleoside triphosphates and Mg(2+). Oligo(A) synthesis was demonstrated with complete virions and infectious subviral particles derived from virus by limited chymotrypsin digestion but not with cores, a product of extensive chymotrypsin digestion of virus. The enzymatic product and the oligo(A) from purified virions were isolated by binding to oligo(dT)-cellulose columns. Most of the in vitro product was similar in size and structure to the oligo(A) from purified virions by the criteria of gel electrophoresis, DEAE-cellulose chromatography, end-group analysis, and sensitivity to RNase. The evidence suggests that oligo(A) synthesis is mediated by the poly(A) polymerase during a late step in viral morphogenesis and may result from an alternative activity of the virion-associated transcriptase.

Adenosine Triphosphate↗
PubMed1972

Capsid polypeptides of mouse Elberfeld virus. I. Amino acid compositions and molar ratios in the virion.

The four major polypeptide chains (alpha, beta, gamma, delta) constituting the capsid protein of mouse Elberfeld (ME) virus were isolated by preparative electrophoresis on polyacrylamide gels, and the amino acid composition of each chain was determined. In addition, the molecular weights of the smallest chains of ME virus, mengovirus, and poliovirus, which had previously been determined by gel electrophoretic methods, were redetermined by gel filtration chromatography in 6 m guanidine hydrochloride. Each was found to have a molecular weight about 7,300. Using the reevaluated molecular weights and the known amino acid compositions of the chains, the molar ratio of each chain in the ME virion was determined by quantitative analysis of the distribution of radioactivity in the electrophoretically separated chains of virus which had been specifically radiolabeled with leucine or with methionine. Equimolar proportions of all four chains were found in the virion.

Amino Acids↗
PubMed1971

Virus-specific proteins synthesized in encephalomyocarditis virus-infected HeLa cells.

The in vivo synthesis of encephalomyocarditis-specific proteins was studied by labeling the viral proteins with radioactive amino acids under conditions where host-protein synthesis was almost completely inhibited. To assure recovery of all proteins, intact cells were lysed in hot 1% sodium dodecyl sulfate. These lysates were analyzed by quantitative high-resolution electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This technique allowed the detection and estimation of the molecular weight of 15 virus-specific polypeptides: A, 100,000; B, 90,000; C, 84,000; D, 75,000, D1, 65,000; E, 56,000; epsilon, 40,000; F, 38,000; alpha, 34,000; beta, 30,000; gamma, 23,000; G, 16,000; H, 12,000; I, 11,000; and delta, 9,000. Pulse-chase experiments, in conjunction with cyanogen bromide and tryptic mapping of the isolated polypeptides, indicate that at least three primary gene products (A,F,C), with a cumulative weight of about 220,000, are generated during translation of the RNA genome. Chains A and C then undergo post-translational cleavages, while F remains uncleaved. The proteins generated by the cleavage of A include all of the capsid chains (alpha, beta, gamma, delta, epsilon). Those generated by the cleavage of C include D and E. The chains alpha, beta, gamma, delta, E, F, G, H, I, with a cumulative molecular weight of about 230,000, are stable and are produced in about equimolar amounts. A model for the synthesis of, and a cleavage sequence that accounts for, all of the viral polypeptides is proposed.

Amino Acids↗
PubMed1969

The structure of mouse-Elberfeld virus: a model.

It is proposed that the capsid structure of mouse-Elberfeld (ME) virus, a small icosahedral ribonucleic acid containing picornavirus, is determined by 60 identical protein subunits; each of the latter is composed of several non-identical polypeptide chains. Icosahedral symmetry in a 60-subunit shell allows three types of specific intersubunit bonding contacts which establish its axes of two-, three-, and fivefold symmetry, respectively. Of these three bonding types, two are sufficient to specify a complete shell. We interpret the possible identity of some discrete supramolecular structures involved in the biosynthesis of poliovirus and in the degradation of ME virus in the context of stepwise formation or disruption of two different types of specific intersubunit bonding contacts.

Electrophoresis↗