L. Andrew Ball

Induction of 2′5′-oligoadenylate synthetase activity and a new protein by chick interferon

Abstract Treatment of primary cultures of chick embryo cells with homologous interferon increased the activity of 2′5′-oligoadenylate synthetase in their cytoplasmic extracts. This enzyme is activated by double-stranded RNA to synthesize the novel trinucleotide pppA 2′ p 5′ A 2′ p 5′ A (and higher oligomers) which is a potent inhibitor of cell-free protein synthesis. The increase in enzyme activity was dependent upon the concentration of interferon and the time of treatment. For example, 1 unit of interferon elicited a 10-fold increase in 20 hr, whereas 100 units or more produced a 2000- to 10,000-fold increase. The kinetics of enhancement showed an initial lag of about 1 hr, followed by a 4- to 5-hr period during which the activity increased exponentially. Maximum levels were reached about 10 hr after interferon treatment. Neither an increase in enzyme activity, nor the establishment of a virus-resistant state was observed in cells treated with chick interferon in the presence of actinomycin D or p -fluorophenylalanine, or in cells treated with heterologous (mouse) interferon. Analysis of the proteins labeled by incorporation of [ 35 S]methionine after interferon treatment revealed the appearance of a new polypeptide in the postribosomal supernatants of interferon-treated cells. Upon gel filtration, this polypeptide coeluted with the peak of oligoadenylate synthetase activity, at 50,000–60,000 MW. It had an apparent molecular weight of 56,000 when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Like the 2′5′-oligoadenylate synthetase activity, the 56,000-dalton polypeptide bound to polyriboinosinic acid -polyribocytidylic acid covalently attached to agarose and could be partially purified in this manner. The appearance of the polypeptide was dependent on the concentration of interferon and the time of treatment employed and was blocked by the presence of actinomycin D. Pulse-labeling studies showed that its rate of synthesis increased sharply about 2 hr after interferon treatment but decreased again about 8 hr later despite the continued presence of interferon in the medium. The possibility that the 56,000-dalton polypeptide is the oligoadenylate synthetase enzyme is being studied. Whatever the outcome, these data suggest that the enzyme and the polypeptide are each the product of a gene whose function is controlled by interferon treatment.

Nuclease activation by double-stranded RNA and by 2′,5′-oligoadenylate in extracts of interferon-treated chick cells

Abstract Cell-free extracts were prepared from primary chick embryo cells that had been pretreated overnight with chick interferon or with mock preparations of interferon. After the addition of double-stranded RNA (dsRNA) to these extracts, the synthesis of vesicular stomatitis virus (VSV) messenger RNA and its corresponding proteins was measured under conditions of coupled transcription and translation directed by purified virus. The addition of dsRNA to extracts of mock interferon-treated cells did not affect VSV-directed transcription or translation. However, in extracts of interferon-treated cells, the rate of RNA accumulation was inhibited by about 50% and that of the viral proteins by about 95% relative to the rates in control extracts. Furthermore, no intact VSV mRNA was detectable among the transcription products made in extracts of interferon-treated cells under these conditions. These results contrasted with those obtained in the absence of dsRNA, where the interferon-mediated inhibition of protein synthesis was less extreme, and where both sets of transcription products were intact and fully functional. The differences could be ascribed, at least in part, to a dsRNA-mediated increase in the rate of mRNA degradation that occurred only in extracts of interferon-treated cells. Enhancement of mRNA breakdown was also observed when protein synthesis was inhibited by the addition of pppA 2′ p 5′ A 2′ p 5′ A and higher oligomers (2′,5′-oligoadenylate) to extracts of either interferon-treated or mock interferon-treated cells. (These novel oligonucleotides are synthesized in interferon-treated cell extracts by an enzyme that requires dsRNA for activation.) Under a variety of conditions, enhanced mRNA degradation correlated with the inhibition of protein synthesis mediated by 2′,5′-oligoadenylate but not by other inhibitors. Thus, the data suggest that the inhibition of protein synthesis by dsRNA in extracts of interferon-treated chick cells is due, at least in part, to mRNA breakdown catalyzed by a nuclease that is activated by 2′,5′-oligoadenylate.

Effect of interferon pretreatment on coupled transcription and translation in cell-free extracts of primary chick embryo cells

Abstract The mechanism of action of chick interferon was studied by the use of cell-free extracts of interferon-treated and mock-interferon-treated primary chick embryo cells. These cell-free extracts differed from those of chick origin which have been used previously in that they did not require supplementation with heterologous factor preparations. In extracts of cells which had been pretreated with mock preparations of interferon, purified vesicular stomatitis virus (VSV) directed the coupled transcription and translation of the viral genes. In extracts of interferon-treated cells, viral transcription was not inhibited, but the rate of translation was reduced by up to 70%, the extent of inhibition being dependent upon the interferon dose. The translation inhibition was not consistently reversed by the addition of chick cell transfer RNA, but was consistently accentuated in the presence of low concentrations of double-stranded RNA. To explore the possibility that this inhibition of protein synthesis was due to an effect on viral messenger RNA synthesis or processing, the products of VSV transcription in the two systems were compared for: (i) their size distribution, by polyacrylamide-gel electrophoresis under denaturing conditions; (ii) their stability, by UTP pulse-chase methods; (iii) their degree of polyadenylation, by oligo(dT)-cellulose column chromatography; (iv) their degree of methylation, by S-adenosyl[ methyl - 3 H]methionine/[ 14 C]UTP double-labeling; and (v) their ability to be translated upon reintroduction into cell-free systems from mock- or interferon-treated chick cells. These tests showed no difference between the preparations of viral mRNA made in the two systems, which indicated that translation itself was the site of inhibition under these conditions. Consistent with this conclusion was the fact that the translation of other messenger RNAs of both viral and animal cell origin was inhibited to a similar extent.

Coupled transcription and translation in mammalian and avian cell-free systems.

Abstract In cell-free extracts of mouse L cells and of primary chick embryo cells, purified vesicular stomatitis virus (VSV) directed coupled transcription and translation of the viral genes. The proportions of virus and cell extract were equivalent to a multiplicity of infection of about 20 PFU per cell. The reactions, which continued for at least 8 hr, resulted in the synthesis of polypeptides which resembled the authentic viral polypeptides N, NS, and M by the criteria of their sizes and tryptic peptide fingerprints. In addition, a minor protein product of the cell-free systems yielded tryptic peptides characteristic of the viral G protein, although its apparent size was slightly less than that of authentic G protein. No product corresponding to the viral L protein was detected. The RNA products of the coupled system were characterized by affinity chromatography on oligo(dT)-cellulose and by polyacrylamide-gel electrophoresis under denaturing conditions. Four size classes of poly(A)-containing RNA were detected, with apparent molecular weights of 2.15, 0.74, 0.55, and 0.3 × 10 6 . These contained the messenger RNAs for the viral L, G, N, and NS + M proteins, respectively. At later times during transcription in vitro , synthesis occurred of increasing amounts of four species of RNA which lacked poly(A), and which had correspondingly lower apparent molecular weights. These species were synthesized de novo , rather than by cleavage of the poly (A) tracts from the RNA made at early times. No RNA corresponding in size to the viral genome was synthesized. The remarkable longevity of the cell-free protein synthesis prompted a study of the structural and functional stability of the VSV messenger RNAs synthesized in the L-cell system. Chase experiments showed that the poly(A)-containing and poly(A)-lacking RNA species were degraded at approximately the same rate, with half-lives of about 12 hr. The methyl groups in the 5′-terminal cap structures of the poly(A)-containing messenger RNAs had a similar half-life. Moreover, preformed VSV messenger RNA added exogenously to the cell-free system at the same concentration as in the coupled system showed a similar rate of degradation. The functional stability of the messenger RNA was investigated by assaying protein synthesis after the addition of S-adenosylhomocysteine to the coupled system to inhibit the synthesis of further translatable messenger RNA. The results showed that the pronounced stability of messenger RNA at low concentration, rather than its continued synthesis, accounted for the longevity of protein synthesis in this system.

INDUCTION, PURIFICATION, AND PROPERTIES OF 2′5′-OLIGOADENYLATE SYNTHETASE*

Treatment of chick embryo cells with interferon causes a dramatic increase in their level of 2′5′ oligoadenylate (2–5A) synthetase activity. Upon activation by double-stranded RNA, this enzyme uses ATP to synthesize a series of oligonucleotides with the general structure (p)ppA 2 ′ (p 5 ′ A) n . Analysis of the polypeptides synthesized in interferon-treated cells reveals the appearance of a new species with an apparent molecular weight of 56,000. This polypeptide is a good candidate for the 2–5A synthetase since it is a major component of partially purified enzyme preparations. Moreover, the 56,000 dalton polypeptide and the synthetase share the following properties: affinity for double-stranded RNA, apparent molecular weight under non-denaturing conditions (50–60,000 MW), elution from DEAE-cellulose colums at 0.05 M KCl, and precipitability with methanol and at pH 5.0.

A TRANSCRIPTIONAL MAP OF VESICULAR STOMATITIS VIRUS

ABSTRACT. Ultra-violet irradiation of nucleic acids can be used to determine the relative sizes of transcriptional units since transcription is blocked beyond the site of a UV hit. We have applied this method to vesicular stomatitis virus by observing the effect of UV radiation on the expression of the viral genes. Gene expression was measured in a cell-free system in which detergent-activated VSV directed coupled transcription and translation. Analysis of the RNA products of this system revealed the expression of the genes for the viral proteins L, G and N; the messenger RNAs for the viral proteins M and NS were not separated by the methods we used. On the other hand, analysis of the protein products revealed the synthesis of the N, NS and M proteins and of a putative precursor to the G protein; no synthesis of the L protein could be detected. Using these two methods, therefore, the expression of each of the five known viral genes could be measured independently.