Graduation date: 2007
The Baculoviridae comprise a diverse group of occluded DNA viruses that contain large double-stranded DNA genomes of 80 - 180 kb and may encode up to 180 gene products. To understand how baculoviruses replicate and process their genomes and the gene products that are involved in these events, a series of mutant virus constructs were generated using the Autographa californica multinucleopolyhedrovirus (AcMNPV) genome propagated as a bacteria artificial chromosome (bacmid). The gene products investigated were very late expression factor-1 (VLF-1), DNA polymerase, alkaline nuclease, the single-stranded DNA binding protein (DBP), and those encoded from the open reading frames, Ac 101, Ac 142, and Ac 144. Using the Spodoptera frugiperda cell line as a tissue culture model system, bacmid constructs lacking any of the open reading frames encoding these seven gene products were shown to be non-infectious. In addition, based on a series of assays to determine the cause for the non-infectious phenotype, it was revealed that VLF-1, a putative recombinase and transcriptional activator was also an essential structural component of the capsid and appeared to possess an enzymatic function related to its role as a recombinase during the final stages of DNA encapsidation. A bacmid lacking the alkaline nuclease gene was able to replicate DNA to normal levels, however, this construct produced aberrant genomes and defective nucleocapsids, suggesting that alkaline nuclease was involved in processing viral genomes during replication. Similarly, analysis of a bacmid lacking the single-stranded DNA binding protein DBP revealed that, although not absolutely required for DNA synthesis, DNA synthesis was reduced in the absence of DBP and no unit-length genomes could be detected from transfected cell extracts. Moreover, although not a structural component of budded virus, the DBP knockout was deficient in nucleocapsid production which is predicted to result from a defect in genome maturation. Finally, the gene products encoded by open reading frames Ac 101, Ac 142, and Ac144, while expendable for DNA synthesis, were shown to be components of the nucleocapsid from budded virus, and bacmids lacking these gene products were deficient in nucleocapsid production.