Germiston virus transcriptase requires active 40S ribosomal subunits and utilizes capped cellular RNAs

Abstract

The transcriptase associated with Germiston virus was assayed in an in vitro reaction in which transcription was coupled to translation by adding reticulocyte lysate under the appropriate salt conditions. When analyzed in polyacrylamide gels, the major transcripts migrated like authentic S mRNAs and possessed 12- to 18-base-long nontemplated 5' extensions similar to the 5' end of viral mRNAs. These transcripts were functional for the synthesis of at least proteins N and NSS. When translation was inhibited by adding protein synthesis inhibitors such as puromycin, cycloheximide, and anisomycin, a drastic inhibitory effect was observed on the synthesis of the complete S mRNA transcripts. However, initiation and part of the elongation process were still active, since short and incomplete RNA molecules with RNA primers at their 5' ends were synthesized. On the other hand, we found that edeine, another inhibitor of protein synthesis, stimulated not only synthesis of S mRNAs but also that of the full-length S cRNAs. Taking into account the mode of action of this antibiotic, we discuss the results, which emphasize the crucial role of active ribosomes during bunyavirus transcription and confirm the observations reported on La Crosse virions. Moreover, we showed that the RNA transcripts synthesized in a transcription-translation reaction were capped and that most of them have acquired the 5' terminal sequences of the alpha- or beta-globin mRNA.