Identification of spliced baculovirus RNAs expressed late in infection

Previous to this study, the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was known to express only one spliced RNA (spliced IE1 or IE0). We have conducted an analysis of RNA expressed during infection of Spodoptera frugiperda cells with AcMNPV and have identified a set of five additional spliced RNAs expressed late in infection. A reverse transcription-polymerase chain reaction analysis was used to confirm the identification of the LS (late, spliced) RNAs. S1 nuclease and primer extension analyses were used to map the transcription initiation sites of LS RNAs. LS1 and LS2 initiated at positions −138 and −117, respectively (relative to the IE0+1 transcription start site). Both LS1 and LS2 contain an additional cistron potentially encoding a small, highly basic polypeptide. LS3 (−79), LS4 (−22), and LS5 (+51/52) RNAs encode only the predicted downstream IE0 ORF. Although several baculovirus late gene consensus transcription initiation sites (ATAAG) were identified within this region, only LS5 initiated at one of these conserved motifs. An S1 nuclease analysis was done to determine whether unspliced precursors of LS RNAs could be detected. Early in infection, a greater proportion of IE0 RNA was detected in the spliced form; however, during the late phase of infection a significantly greater amount of unspliced precursor forms of LS RNAs was observed.