Synthesis of (2'-5')oligoadenylate and activation of an endoribonuclease in interferon-treated HeLa cells infected with reovirus.

Treatment with interferon protected HeLa cells from infection with reovirus. This virus apparently activated an antiviral mechanism that was detected by the presence of (2'-5')oligoadenylate [(2'-5')An] in intact cells. The (2'-5')An was previously shown to activate an endoribonuclease, RNase L. We measured (2'-5')An by a sensitive competition-binding assay in cells infected at different multiplicities and for different lengths of time. Nanomolar concentrations of (2'-5')An were detected in cells infected at a multiplicity of greater than 5 after 2 h of infection, the time at which the infecting virions were uncoated. The level of (2'-5')An increased up to 6 h postinfection but declined afterward. To establish whether viral mRNAs were cleaved by RNase L, we analyzed the RNA extracted from infected cells by a highly specific hybridization assay on Northern blots. Full-sized reovirus mRNAs were detected in control infected cells, but not in interferon-treated infected cells, at 6 h postinfection. At this time, a nuclease activity could be detected in these cells by demonstration of cleavage of rRNA, degradation of cellular mRNA, and polysome breakdown in the presence of emetine. Since this inhibitor freezes ribosomes, cleavage of mRNA between ribosomes could only be accounted for by an endonuclease, presumably RNase L.     

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.     

Double-stranded RNA causes synthesis of 2',5'-oligo(A) and degradation of messenger RNA in interferon-treated cells

Interferon-treated HeLa cells were incubated with [3H]uridine to label mRNA and were then exposed to the double-stranded RNA poly(inosinic acid).poly(cytidylic acid) (In.Cn). The incubation with In.Cn greatly enhanced the decay of mRNA. When the cells were incubated in this way in the presence of cycloheximide, which blocks ribosome movement along mRNA, extensive polysome degradation was detected in interferon-treated cells. Products of degradation of mRNA were recovered from monosomes which were presumably formed as a result of endonucleolytic breaks of mRNA. This endonucleolytic activity was correlated with the formation of 2',5'-oligo(A) by an enzyme induced by interferon and activated by double-stranded RNA; the 2',5'-oligo(A) was previously shown to activate an endonuclease in cell extracts. The 2',5'-oligo(A) levels in cells were measured by a competition-binding assay. Details of the procedure used are described, including synthesis of highly radioactive (2'-5')pppA3[32P]cytidine 3',5'-diphosphate, separation of 2',5'-oligo(A) binding from degrading activities, and specificity of the assay.     

Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.     

Role of 2',5'-oligo(adenylic acid) polymerase in the degradation of ribonucleic acid linked to double-stranded ribonucleic acid by extracts of interferon-treated cells

RNA covalently linked to double-stranded RNA (dsRNA) is preferentially degraded in extracts of interferon-treated HeLa cells [Nilsen, T. W., & Baglioni, C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2600-2604]. The size of the dsRNA required for this preferential degradation has been determined by annealing poly(I) of known length to the poly(C) tract of encephalomyocarditis virus (EMCV) RNA or by annealing poly(U) to poly(A) of known length of vesicular stomatitis virus mRNA. The dsRNA must be longer than about 60 base pairs to observe the preferential degradation of RNA. Moreover, triple-stranded regions that do not activate synthesis of 2',5'-oligo(A) and ethidium bromide, which intercalates in dsRNA and blocks 2',5'-olido(A) polymerase activation, prevent this degradation. Ethidium also blocks the degradation of the replicative intermediate of EMCV by extracts of interferon-treated cells. These experiments indicate that synthesis of 2',5'-oligo(A) is required for the degradation of RNA linked to dsRNA. The 2',5'-oligo(A)-dependent endonuclease does not cleave single- or double-stranded DNA, nor does it cleave homopolyribonucleotides. The potential role of the 2',5'-oligo(A) polymerase/endonuclease system in the inhibition of viral RNA replication is discussed.     

Mechanism of interferon action. Effect of double-stranded RNA and the 5'-O-monophosphate form of 2',5'-oligoadenylate on the inhibition of reovirus mRNA translation in vitro

The effect of reovirus double-stranded RNA (dsRNA) and 5'-O-monophosphate form of 2',5'-oligoadenylate (pA(2'p5'A)2) on the translation and degradation of reovirus messenger RNA and on protein phosphorylation was examined in extracts prepared from interferon-treated mouse L fibroblasts. The following results were obtained. 1) The enhanced degradation of reovirus [3H]mRNA observed in the presence of either dsRNA or the 5'-O-triphosphate form of 2',5'-oligoadenylate (pppA(2'p5'A)3) was completely blocked by pA(2'p5'A)2. 2) The dsRNA-dependent phosphorylation of protein P1 and the alpha subunit of eukaryotic initiation factor (eIF-2) depended in a similar manner upon the concentration of dsRNA and was optimal at low dsRNA concentrations (0.1 to 1 microgram/ml). However, high concentrations of dsRNA (greater than 100 micrograms/ml) drastically reduced the phosphorylation of both P1 and eIF-2 alpha. Neither P1 nor eIF-2 alpha phosphorylation was affected by either pA(2'p5'A)2 or pppA(2'p5'A)3. 3) The translation of reovirus mRNA in vitro was inhibited by the addition of either low concentrations of dsRNA or pppA(2'p5'A)3. Whereas pA(2'p5'A)2 completely reversed the pppA(2'p5'A)3-mediated inhibition of translation, the inhibition mediated by low concentrations of dsRNA was only partially reversed by pA(2'p5'A)2. Under conditions where the pppA-(2'p5'A)3mediated degradation of reovirus mRNA was blocked, the translation of reovirus mRNA was still inhibited by low but not by high concentrations of dsRNA in a manner that correlated with the activation of P1 and eIF-2 alpha phosphorylation. These results suggest that the pppA(2'p5'A)n-dependent ribonuclease is not required and that protein phosphorylation may indeed be sufficient for the dsRNA-dependent inhibition of reovirus mRNA translation in cell-free systems derived from interferon-treated mouse fibroblasts.     

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes. The four enzyme forms also differ in their intracellular localization, on microsomes (100 kDa), in nuclei (67, 46, 40 kDa), and on membrane structures (67 kDa). Plasma membranes from interferon-treated Daudi lymphoblastoid cells are highly enriched in the 67-kDa 2',5'-oligo(A) synthetase form. The 2',5'-oligo(A) synthetase activity induced by interferons in human cells appears, therefore, as a complex multienzyme system.     

Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.     

The 2'-5' oligoadenylate/RNase L/RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation

The 2'-5' oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.     

Involvement of the interferon-regulated antiviral proteins PKR and RNase L in reovirus-induced shutoff of cellular translation

Cellular translation is inhibited following infection with most strains of reovirus, but the mechanisms responsible for this phenomenon remain to be elucidated. The extent of host shutoff varies in a strain-dependent manner; infection with the majority of strains leads to strong host shutoff, while infection with strain Dearing results in minimal inhibition of cellular translation. A genetic study with reassortant viruses and subsequent biochemical analyses led to the hypothesis that the interferon-induced, double-stranded RNA-activated protein kinase, PKR, is responsible for reovirus-induced host shutoff. To directly determine whether PKR is responsible for reovirus-induced host shutoff, we used a panel of reovirus strains and mouse embryo fibroblasts derived from knockout mice. This approach revealed that PKR contributes to but is not wholly responsible for reovirus-induced host shutoff. Studies with cells lacking RNase L, the endoribonuclease component of the interferon-regulated 2',5'-oligoadenylate synthetase-RNase L system, demonstrated that RNase L also down-regulates cellular protein synthesis in reovirus-infected cells. In many viral systems, PKR and RNase L have well-characterized antiviral functions. An analysis of reovirus replication in cells lacking these molecules indicated that, while they contributed to host shutoff, neither PKR nor RNase L exerted an antiviral effect on reovirus growth. In fact, some strains of reovirus replicated more efficiently in the presence of PKR and RNase L than in their absence. Data presented in this report illustrate that the inhibition of cellular translation following reovirus infection is complex and involves multiple interferon-regulated gene products. In addition, our results suggest that reovirus has evolved effective mechanisms to avoid the actions of the interferon-stimulated antiviral pathways that include PKR and RNase L and may even benefit from their expression.     

Inhibition of polypeptide chain initiation in Daudi cells by interferons. Evidence that activity of initiation factor eIF-2 and availability of mRNA are unimpaired.

The accompanying paper [McNurlan & Clemens (1986) Biochem. J. 237, 871-876] shows that the inhibition of proliferation of Daudi cells by human interferons is associated with impairment of the overall rate of protein synthesis. We have examined whether two of the mechanisms which are believed to control translation in interferon-treated virus-infected cells may be responsible for the inhibition of protein synthesis during the antiproliferative response in these uninfected cells. Although the rate of polypeptide chain initiation is lower in interferon-treated Daudi cells, as indicated by the disaggregation of polysomes, there is no significant inhibition of activity of initiation factor eIF-2 or of [40 S . Met-tRNAf] initiation complex formation in cell extracts. The phosphorylation state of the alpha subunit of eIF-2 remains unaltered. There is no major decrease in mRNA content as a proportion of total RNA up to 4 days of interferon treatment, as judged by poly(A) content, although the amount of total mRNA/10(6) cells eventually declines. The mRNA present in extracts from interferon-treated cells remains translatable when added to an mRNA-dependent reticulocyte lysate system. We conclude that neither the interferon-inducible eIF-2 protein kinase pathway nor the 2',5'-oligo(adenylate)-ribonuclease L pathway are responsible for the inhibition of polypeptide chain initiation. Rather, the data suggest impairment at the level of formation of [80 S ribosome X mRNA] initiation complexes.     

Presence of 2',5'-oligo(A) and of enzymes that synthesize, bind, and degrade 2',5'-oligo(A) in HeLa cell nuclei

Nuclei prepared from HeLa cells by lysis with nonionic detergents or by a nonaqueous fractionation procedure were assayed for enzymatic activities which synthesize, bind, and degrade 2',5'-oligo(A). Isolated nuclei synthesized micromolar concentrations of 2',5'-oligo(A) when incubated with poly(inosinic) . poly(cytidylic) acid. The products of nuclear synthesis were identified with authentic 2',5'-oligo(A) by several criteria. The nuclei synthesized nanomolar amounts of 2',5'-oligo(A) even when incubated without added double-stranded RNA. These oligonucleotides were identified by their pattern of degradation with different nucleases and by a specific competition-binding assay. This assay revealed the presence in nuclei of an activity which binds 2',5'-oligo(A) with an affinity constant similar to that of the cytoplasmic binding activity previously identified with the 2',5'-oligo(A)-dependent endoribonuclease (Nilsen, T. W., Wood, D. L., and Baglioni, C. (1981) J. Biol. Chem. 256, 10751-10754). The nuclei had also an activity which degraded 2',5'-oligo(A). Finally, unincubated nuclei isolated by the nonaqueous fractionation procedure contained detectable concentrations of 2',5'-oligo(A). These results show that an activator of the enzyme which synthesize 2',5'-oligo(A) is present in nuclei and that these oligonucleotides are normally formed in HeLa cells, and suggest a possible role for the 2',5'-oligo(A)-activated endoribonuclease in nuclear RNA metabolism.     

Effect of RNA synthesis inhibitors on insulin-induced protein synthesis by 3T3 cells

1. The increased protein synthesis of quiescent 3T3 cells in response to insulin was separated into three distinct phases based on their response to various inhibitors of RNA synthesis. 2. The first increase in protein synthesis was insensitive to the inhibitors used, and probably resulted from activation of existing protein synthesizing mechanism. 3. The second phase was sensitive to a varying extent to alpha-amanitin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, implying the need for new mRNA synthesis as well as the production of new ribosomes indicated by its further sensitivity to low concentration (10 ng/ml) of Actinomycin D. 4. The final phase was insensitive to inhibitors of new ribosome formation, but still depended on new mRNA. alpha-difluoromethylornithine, an inhibitor of de novo polyamine synthesis, partly inhibited the insulin induced stimulation of protein synthesis.     

Activation and evasion of the antiviral 2'-5' oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA

Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-alpha2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 2'-5' oligoadenylate synthetase/ ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA.