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

Abstract

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 Ehrlich ascites tumor cells (S30INT). We find now that after the methylation of reo mRNAu has stopped in S301NT, the RNA can be reisolated and further methylated in an extract of control cells (S30C). Thus the impairment of methylation in S301NT cannot be due to cleavage or irreversible inactivation of reo mRNAu. Freshly added reo mRNAu can be methylated in S301NT in which the methylation of previously added reo mRNAu has stopped. This indicates that the impairment is not due to the depletion ofS-adenosylmethionine (the methyl donor), the accumulation of S-adenosylhomocysteine (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 S301NT

Regulation of cell proliferation and differentiation by interferons

“…The protein kinase phosphorylates the a subunit of initiation factor eIF-2, thereby inhibiting the ability of the factor to catalyse the initiation of protein synthesis. I possible modes of inhibition in some infected cell types include interferon-induced impairment of 5'methylation of newly synthesized mRNA (Sen et al, 1977;de Ferra & Baglioni, 1983) and inactivation of the amino acid accepting ability of certain tRNA species (Zilberstein et al, 1976) (Table 1). These effects are not dependent on dsRNA.…”

Metabolic stability of 2′ 5′oligo (A) and activity of 2′ 5′oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells

“…Several investigators have compared biochemical characteristics of extracts prepared from interferon-treated cells with those of extracts from control cells in order to elucidate the molecular basis of these defense mechanisms. Among the many differences reported between these extracts, the most significant appear to be: 1) the enhanced inhibition of protein synthesis by double stranded RNA (dsRNA) (3)(4); 2) the decreased methylation of the 5'-terminal guanosine of added viral mRNA (5)(6); 3) an increase in three enzymatic activities, which are dependent on the addition of dsRNA to cell extracts. These are a protein kinase (7)(8)(9), an endonuclease (10)(11)(12) and an enzyme which utilizes ATP for the synthesis of 2'5'oligo(A), an oligonucleotide with the structure pppA(2'p5'A) (where n is 2 to 6) (13)(14)(15)(16).…”

“…Inhibition of protein synthe-6 _ sis in reticulocyte lysates. Protein synthesis was assayed as previously described(21) in the presence of increasing concentrations of 2'5'oligo(A).E Incorporation is expressed as cpm/5 QI_D ]S/°°pl incubation.…”

RNA methylation in vaccinia virus-infected chick embryo fibroblasts treated with homologous interferon

“…A qualitatively different reduction in ribose methylation leading to a shift from "cap II" to "cap I" structures in interferon-treated reovirus-infected mouse Lcells has been reported in a recent abstract (24). An impairment of reovirus mRNA methylating activity had also been detected in cell-free extracts from Ehrlich Ascites cells pretreated with interferon (25,26). However, it was concluded from these studies, that in order to ascertain the physiological role of these findings, investigations of the mRNA methylation pattern in interferon-treated infected cells were required.…”

“…In order to investigate the functional significance of this interferon-induced change, we have extended the analysis of the methylated nucleotides to transfer and ribosomal RNA isolated from interferon-treated and control chick embryo fibroblasts. The results of our studies using intact vaccinia-infected cells are discussed with respect to the physiological significance of impaired reo-mRNA methylating activities reported from interferon-treated murine cells (25,26).…”