An investigation of Autographa californica multiple nucleopolyhedrovirus DNA replication.

摘要

Different approaches were used to investigate the molecular mechanisms of the type baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA replication. Several DNA replication inhibitors were initially identified as potentially useful reagents for AcMNPV DNA replication studies. To facilitate this, two rapid and sensitive assays based on spectroflurometric analysis of EGFP expressed by a recombinant AcEGFP virus and qPCR analysis were developed. Of the twenty one compounds tested, seven, including aphidicolin, abacavir, camptothecin, E)-5-(2-bromovinyl)-2'-deoxyuridine, L-mimosine, hydroxyurea and phosphonoacetic acid were identified as selective inhibitors of AcMNPV DNA replication as they reduced EGFP expression and viral DNA accumulation, in infected cells. Of these compounds, the inhibitory effects of aphidicolin, abacavir, L-mimosine and hydroxyurea on AcMNPV DNA replication were reversible. Together, both spectrofluorimetric and qPCR assays were judged to be suitable for rapid quantitative investigation of inhibitors of baculovirus DNA replication in infected cells.;Finally, the in vivo DNA ori activity of two structurally heterogenous putative origins of AcMNPV DNA replication (hr5 and the non-hr region) was compared using a nascent DNA abundance assay and qPCR analysis. Preliminary results suggested that at 18 hpi, the oriK exhibited highest levels of nascent DNA molecules and the direction of replication apperaed to proceed bidirectionally from the oriK.;To better understand the baculovirus DNA polymerase, AcMNPV DNA pol drug "escape" mutants were generated and characterized. Two mutants, AcAPCR and AcABCR, were generated in cell culture by serial passage of the parental AcMNPV in the presence of increasing concentrations of the inhibitor aphidicolin and abacavir, respectively. Drug escape mutant characterization showed similar virus genome structure and replication profiles relative to parental virus. However, the mutants exhibited altered occlusion body morphology. Sequence analysis of the dnapol gene of AcAPCR and AcABCR revealed point mutations within the conserved regions III and or II, respectively of the protein but no mutation in other viral DNA replication proteins. Complementation experiments suggested these mutations are responsible for the resistance phenotype against both drugs.