Mechanism of Interferon Action: Inhibition of Viral Messenger Ribonucleic Acid Translation in L-Cell Extracts

Abstract

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of "4C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of "4C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5 IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vacciniainfected cells were smaller than those formed in extracts of untreated, vacciniainfected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.

Antiviral activities of interferons

“…(i) Infection-activation of antiviral action. Friedman et al (1972) found that viral mRNA translation was the same in cell-free extracts prepared from normal and interferon-treated cells; however, extracts prepared from vaccinia or EMC virusinfected interferon-treated cells would not translate viral messages, although polyuridylic acid translation was uninhibited. Kerr et al (1973Kerr et al ( , 1974b then concluded that interferon-induced antiviral action requires virus infection to activate translation inhibition.…”

“…With the advances of interferon purification techniques, recent studies have been performed with interferon preparations with high specific activities. Voluminous information on the ability of interferon to inhibit virus-specific protein synthesis (Joklik and Merigan, 1966;Salb, 1966, 1968;Carter and Levy, 1967a, b;Levy and Carter, 1968;Kerr et al, 1970;Kerr, 1971;Esteban and Metz, 1973;Metz and Esteban, 19;/2;Friedman, 1968;Friedman et al, 1972) indicates clearly that at least one of the mechanisms of interferon actions is at the translational level. Actions of interferon at the transcriptional level have also been reported, with inhibition of viral mRNA synthesis in polymerase-containing viruses (Marcus et al, 1971;Oxman and Levin, 1971;Bialy and Colby, 1972;Manders et al, 1972), perhaps through an inhibition of the viral polymerase function.…”

Influence of interferon on the synthesis of virus particles in oncornavirus carrier cell lines. III. Survey of effects on A‐, B‐ and C‐type oncornaviruses

“…Conceivably, the synthesis of non-infectious oncornaviruses is governed by an incorporated provirus, while infectious viruses are formed by repeated infectious cycles. Although the mechanism of interferon action is still a matter of conjecture (Marcus and Salb, 1966;Levy and Carter, 1968;Kerr, 1971; Marcus et al, 1971; Metz and Esteban, 1972;Bialy and Colby, 1972;Falcoff et al, 1972;Friedman et al, 1972;Gupta et al, 1974;Samuel and Joklik, 1974) it is generally accepted that interferon treatment inhibits the transcription or translation of exogenous viral genetic information to a greater extent than that of endogenous cellular information. In accordance with this concept, one might predict that the synthesis of provirusdirected virions would be relatively resistant to interferon.…”

Intracellular Events in Interferon-Treated Cells

“…Furthermore, such tRNA inactivation was also dependent on preincubation of the cell extracts [381. Therefore, the relevance of the tRNA reversible inhibition is questionable since it develops during preparation of the cell-free system and, moreover, tRNA isolated from interferon-treated cells shows the same ability cells with interferon as a necessary step for the manifestation of the interferon-mediated inhibition of translation [47]. The translation of EMC RNA in cell-free systems from mouse L-cells is well studied [30, 351.…”