Cross-linking of viral RNA by 4'-aminomethyl-4,5',8-trimethylpsoralen in HeLa cells infected with encephalomyocarditis virus and the tsG114 mutant of vesicular stomatitis virus

The presence of double-stranded RNA (dsRNA) of viral origin in infected cells is controversial. We established that in intact HeLa cells infected with encephalomyocarditis virus (EMCV) viral RNA can be cross-linked with 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT). This compound intercalates into dsRNA and forms cross-links upon irradiation with uv light. Addition of AMT to EMCV-infected cells, followed by irradiation, resulted in a dose-dependent inhibition of viral RNA synthesis, This inhibition was correlated with the cross-linking of nascent RNA strands to template strands of the viral replicative intermediate of EMCV. In contrast, treatment with AMT of vesicular stomatitis virus (VSV)-infected cells had no effect on viral mRNA synthesis and no cross-linking of viral RNA could be detected. The synthesis of viral RNA by the tsG114 mutant of VSV at the nonpermissive temperature, however, was inhibited by AMT. In cells infected with this mutant, cross-linked viral RNA could be detected. These results are discussed with regard to the role of dsRNA in cellular responses to viral infection mediated by interferon.