Molecular dissection of the Spodoptera littoralis nucleopolyhedrovirus: Virus-host cell interaction and virus DNA replication.

摘要

Baculoviruses are viruses of arthropods with large rod-shaped virions that contain supercoiled double-stranded DNA genomes. The mechanisms of baculovirus DNA replication and virus-host interaction are still poorly understood. I studied the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV). Previous investigations indicated that SpliNPV possesses a unique host-range and genetic organization. I studied the SpliNPV infection of an orthopteran cell line derived from the grasshopper, Melanopus sanguinipes , and provided evidence of viral DNA replication and production of viable virus progeny. I next investigated SpliNPV infection in five cell lines derived from three lepidopteran families: Sf9, CLS79 and Se1 cell lines from Spodoptera (Noctuidae), Ld652Y cells from Lymantria dispar (Lymantriidae), and Md210 cells from Malacosoma disstria (Lasiocampidae), which represented permissive (Sf9, CLS79, and Se1), semi-permissive (Ld652Y), and non-permissive (Md210) cell lines. SpliNPV infection in permissive cell lines resulted in viral gene expression, DNA replication, and production of viable progeny. While the semi-permissive cell line displayed reduced and delayed transcription of viral genes and supported limited viral DNA replication, the non-permissive cell line displayed dramatically reduced viral transcription and abolished viral progeny. Transient expression assays using SpliNPV early- or very late-promoter reporters suggested that non-productive infection of SpliNPV in semi- or non-permissive cell lines was a consequence of limited viral specific transcription at the early phase of viral infection.;I investigated the mechanism of SpliNPV DNA replication. Using transient replication assays I have identified a non-hr origin of SpliNPV DNA replication. The putative SpliNPV origin consists of sequence motifs found in other origins of virus DNA replication. Transient expression assays indicated that the putative non-hr origin represses the SpliNPV early gene, lef-3. Gel mobility shift analyses confirmed that nuclear proteins from both infected and uninfected cells bound with specificity to the putative origin.;I then identified a trans-acting factor involved in viral DNA replication. I have sequenced a 6.4 kb DNA from SpliNPV genome that contains an ORF encoding a predicated polypeptide of 998 amino acid sequences. Comparative sequence analyses demonstrated that the ORF encoded a DNA polymerase (dnapol) that consists of conserved exonuclease domains and DNA polymerase motifs found in other eukaryotic DNA viruses. (Abstract shortened by UMI.)