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P Lengyel

Publications and source records attributed to P Lengyel.

15 recordsLinked to original sources
PubMed1979

Structural characteristics of interferons from mouse Ehrlich ascites tumor cells.

An improved procedure for the isolation of interferons produced by mouse Ehrlich ascites tumor cells infected with Newcastle disease virus provides interferons of three size classes (33,000, 26,000, and 20,000 daltons) with specific activities between 2 and 3 x 10(9) units/mg of protein and a yield of 11 to 20%. The tryptic peptide maps of the two larger species are very similar; that of the smallest species is different, at least in part. The amino acid compositions of the three species are very close. Their NH2-terminal amino acids are identical and so are the amino acids released by carboxypeptidase A treatment. These data are consistent with the possibility that the differences in size between the three species may be due, at least in part, to unequal glycosylation.

Amino Acids↗
PubMed1979

Impairment of reovirus mRNA 'cap' methylation in interferon-treated mouse L929 cells.

Reovirus mRNAs synthesized in vitro by the virionassociated enzyme have a 5' 'cap 1' structure (m7G(5')ppp(5')GmpCp...). However, about one third to one half of the reovirus mRNAs formed in mouse L929 cells have a 5' 'cap 2' structure (m7G(5')ppp(5')GmpCmp...) and the rest have a 5' 'cap 1' structure. The finding that virus mRNA 'cap' methylation is impaired in extracts of interferon-treated cells prompted us to study the effect of interferon on virus mRNA 'cap' methylation in vivo. Using labeling with [3H]-guanosine and dual labeling with [3H]methionine and [14C]uridine we compared the 5' structures of reovirus mRNAs accumulating between 5 and 11 h after infection in: L929 cells treated with 390 to 2600 U/ml of a partially purified mouse interferon preparation and untreated L929 cells. The treatment resulted in a 70 to 98% decrease in the 24 h virus yield and in a 50 to 55% decrease in the label accumulated in virus mRNAs. The 'capping' of virus mRNAs and the methylation of their 5' terminal and adjacent G residues were not diminished in interferon-treated cells. However, the percent of 'cap 2' termini was 36 to 47% lower in virus mRNAs from interferon-treated cells than in virus mRNAs from control cells. The interferon treatment did not result in the appearance of additional methylated nucleotides in the virus mRNAs.

Animals↗
PubMed1979

Interferon, double-stranded RNA, and RNA degradation: activation of an endonuclease by (2'-5')An.

Among the mediators of interferon action are one enzyme that is activated by double-stranded RNA to convert ATP to (2'-5')An and a second enzyme, an endonuclease, that is activated by (2'-5')An to cleave single-stranded RNA. The binding of (2'-5')An to the endonuclease (partially purified from mouse Ehrlich ascites tumor cells) is revealed by its retention on nitrocellulose filters. This can serve as the basis for an assay of the enzyme. Activation of the enzyme is reversible and is lost upon removal of (2'-5')An:gel filtration of activated endonuclease on Sephacryl S-200 results in an inactive enzyme. The enzyme can be activated again, however, by addition of (2'-5')An. The elution volume of the nonactivated endonuclease from Sephadex G-200 indicates that its molecular weight is 185,000, unusually large for a nuclease. The elution volume of the maximally activated endonuclease from Sephadex G-200 equilibrated with (2'-5')An is not detectably different from that of enzyme that had not been previously activated that was passed through Sephadex G-200 not equilibrated with (2'-5')An. This indicates that the activation does not result in a large change in the size or conformation of the enzyme.

Animals↗
PubMed1978

Purification of interferon from mouse Ehrlich ascites tumor cells.

Interferon production was induced in mouse Ehrlich ascites tumor cells by infection with Newcastle disease virus. The interferon produced was purified by precipitation with ammonium sulfate, chromatography on carboxymethyl-Sephadex, treatment with blue dextran and polyethylene glycol, gel filtration on Bio-Gel P-60 and Bio-Gel P-200, chromatography on phosphocellulose, isoelectric focusing, and chromatography on octyl-Sepharose. The specific activity of the product was 1.6 x 10(9) NIH mouse interferon reference standard units/mg of protein. Electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate indicated that the apparent molecular weight of the interferon-active material ranged from 25,000 to 35,000. As revealed by staining the gels with Coomassie brilliant blue, the interferon activity co-migrated with the major, broad protein band. Minor, stainable bands of proteins were free of interferon activity and their apparent molecular weight was smaller than 12,000.

Animals↗
PubMed1978

Interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded RNA.

Double-stranded RNA inhibits protein synthesis in at least two ways. It activates a protein kinase that blocks peptide chain initiation by phosphorylating the peptide chain initiation factor eIF-2 and also activates an endonuclease that inactivates different mRNAs at different rates. The protein kinase and the endonuclease have been partially purified from interferon-treated Ehrlich ascites tumor cells. The 2',5'-oligoadenylates [pppA(2'p5'A)n], found found earlier to be mediators in the activation of the endonuclease by double-stranded RNA, are not mediators in the activation of the protein kinase by double-stranded RNA.

Adenine Nucleotides↗
PubMed1977

Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrilich ascites tumor cells: further characteristics of the phenomenon.

We reported earlier that the methylation of unmethylated reovirus mRNA (reo mRNAU) by the cellular methylating enzymes is impaired in extracts of uninfected, interferon-treated Ehrilich ascites tumor cells (S30INT). We find now that after the methylation of reo mRNAU has stopped in S30INT, the RNA can be reisolated and further methylated in an extract of control cells (S30C). Thus the impairment of methylation in S30INT cannot be due to cleavage or irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is due to the depletion of S-adenosylme thionine (the methyl donor), the accumulation of S-adenosylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is not due to the depletion of S-adenosylmethionine (the methyl donor), the accumulation of S-adenoxylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of the methylating enzymes. It may be due, however, to the unavailability of reo mRNAU for methylation. The extent of the impairment of reo mRNAU methylation in S30INT decreases with an increasing concentration of reo mRNAU but is not affected by added poly (U), ribosomal RNA, or encephalomyocarditis virus RNA (an mRNA that is probably not capped or methylated at its 5' end). The methylation of reo mRNAU is also impaired in an extract from cells that have not been treated with interferon but with the interferon inducer poly(I) - poly(C). The inhibitor is apparently a macromolecule that is inactivated during incubation. It decreases the methylation at the 7 position of the 5' terminal guanylate residue. In vitro, the rate of reo mRNA synthesis by reovirus cores in the presence of S30INT is the same as in the presence of S30C. However, the methylation of the de novo synthesized reo mRNA by the core-associated methylating enzyme(s) in vitro is inhibited by S30INT but not by S30C. The relevance of these phenomena to the inhibition of reovirus replication in interferon-treated cells remains to be established.

Animals↗
PubMed1977

Messenger RNA methylation, translation and degradation in extracts of interferon-treated cells.

Extracts from interferon-treated, not virus infected EAT cells differ in several biochemical characteristics from extracts of untreated cells. Some of these differences are manifested only if the extracts are supplemented with ds RNA and ATP. Thus, in the extracts from interferon-treated cells these supplements activate a protein kinase and an endonuclease activity as well as an inhibitor of the translation of messenger RNA. The effect of the same supplements in extracts of untreated cells is much less pronounced. Other differences between the two types of extracts do not seem to depend on the addition of ds RNA and ATP. These include an impairment of mRNA cap methylation and an inhibition of peptide chain elongation that can be overcome by the addition of tRNA. The treatment of human (HeLa S3) cells with human interferon is manifested in the cell extract similarly to the treatment of EAT cells with mouse interferon. Studies are underway to isolate and characterize the ds RNA activated enzymes and the inhibitors and to establish how the presence of these in extracts from interferon-treated cells can account for the impairment of virus replication by interferon.

Adenosine Triphosphate↗
PubMed1976

Formation and characteristics of reovirus subviral particles in interferon-treated mouse L cells.

We examined the early phase of reovirus replication in interferon-treated L cells. No difference was detected in the adsorption of virus to cells, the rate and extent of conversion of parental virions to sub-viral particles (SVPs), and the protein and double-stranded RNA composition of the SVPs when comparing reovirus infection of the interferon-treated and control cells. Furthermore, when tested in vitro, SVPs isolated from interferon-treated cells (SVPINT) synthesized and methylated reo mRNAs at the same rate as SVPs isolated from control cells (SVPCON). However, the accumulated products of RNA synthesis promoted by SVPCON consisted mainly of full size reo mRNA molecules, whereas those whose synthesis was promoted by SVPINT consisted mainly of shorter products. These results indicate that premature termination of transcription and/or degradation of full size transcripts occurred in vitro with SVPINT. Other experiments revealed that a nuclease is associated with our SVPINT preparation.

Animals↗
PubMed1975

Interferon treatment of Ehrlich ascites tumor cells: effects on exogenous mRNA translation and tRNA inactivation in the cell extract.

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S60INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30INT between tRNA from interferon-treated cells and tRNA from control cells. Futhermore, no difference was found in the rate of inactivation in S30INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under out conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation fo exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.

Animals↗