Transcription of overlapping sets of RNAs from the genome of Autographa californica nuclear polyhedrosis virus: a novel method for mapping RNAs

Abstract

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) contains a double-stranded, supercoiled circular genome of 126 to 129 kilobase pairs in length. In cultured Spodoptera frugiperda insect cells the virus replications, and early and late phases of viral genome expression are discernible. We previously mapped 5 early and at least 32 late different viral polypeptides on the viral genome (H. Esche, H. Lübbert, B. Siegmann, and W. Doerfler, EMBO J. 1:1629-1633, 1982). However, at the same time we located 11 early and more than 90 late different size-classes of viral RNA on the AcNPV genome. Evidence for extensive RNA splicing in this virus system has not been adduced, although tiny splices cannot yet be ruled out (H. Lübbert and W. Doerfler, J. Virol. 50:497-506, 1984). The large number of AcNPV transcripts and the apparent lack of splicing have raised tantalizing questions about the mechanisms involved in the expression of AcNPV DNA and its regulation. It is also unknown how the widely differing numbers of RNAs and polypeptides can be correlated. For this reason, we have started to analyze in detail the map locations of some of the RNA size-classes in three different segments of the viral genome. For this purpose a novel method has been devised which will prove useful for the analyses of transcriptional patterns in complex viral genomes. The EcoRI fragments J, O through F, and Q, comprising viral DNA segments between 81.8 and 86.4, 32.6 and 41.0, and 88.2 and 89.7 map units, respectively, were investigated. Surprisingly, overlapping sets of viral RNAs of various lengths and with apparently common 3' termini in EcoRI fragments J (seven size-classes) and O through F (four size-classes) or with common 5' termini in EcoRI fragment Q (two size-classes) have been detected. At present, the functional significance of this mode of transcription is unknown. EcoRI fragment Q of AcNPV DNA encodes a 10,000-molecular-weight polypeptide which is expressed abundantly late after infection. The function of this protein has not yet been elucidated. The promoter and 5' part of the gene for the 10,000-molecular-weight polypeptide have been sequenced, and we have shown that at least two RNAs of different lengths are transcribed in this region and initiated at one site of three nucleotides. Studies on the expression of the AcNPV genome have revealed interesting properties not commonly found in other eucaryotic systems.