Functional aspects of baculovirus DNA photolyases

Keywords: baculovirus, ChchNPV, CPD photolyase, phylogeny, UV resistance, DNA binding, localization, proteomics

Baculoviruses are insect viruses that are applied as biological control agents due to adequate virulence, host specificity and safety for the environment. Solar light negatively affects field performance of baculoviruses by reducing their infectivity, most likely as a consequence of the formation of cyclobutane pyrimidine dimers (CPDs) in the viral DNA upon ultraviolet (UV) irradiation. CPDs can be repaired by CPD photolyases when exposed to blue light photons, a process called photoreactivation. From previous work it was known that the Cc-phr2 gene of the baculovirus Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) encodes a biochemically active photolyase. The research in this thesis focuses on (i) the degree of conservation of CPD photolyase (phr) genes in a subgroup of baculoviruses, (ii) the localization of baculovirus photolyase proteins in insect cells and occlusion derived virus (ODV), and (iii) the in vivo effect of phr genes on the UV sensitivity of baculoviruses. Homologues of the Cc-phr genes were found in all studied group II NPVs in the genus Alphabaculovirus that infect insects in the subfamily Plusiinae insects. Phylogenetic analysis suggested that these phr-like genes have a common ancestor. Intracellular localization of the two ChchNPV encoded PHR proteins in insect cells was studied using enhanced GFP fusion. Both PHR1 and PHR2 localized in the nucleus and associated with chromosomes, spindle, aster and midbody structures during host cell mitosis. Moreover, Cc-PHR2 co-localized with virogenic stroma, when PHR2-EGFP-transfected cells were infected with Autographa californica (Ac) MNPV. Neither of the two Cc-PHR proteins was identified by LC/MS-MS in the ODVs of ChchNPV. To evaluate the potential of the Cc-PHR2 protein to reduce the UV sensitivity of a baculovirus, the Cc-phr2 gene was incorporated in the genome of Helicoverpa armigera (Hear) NPV, which does not have a UV damage repair system. This resulted in a decreased sensitivity to UV-light compared to wild type HearNPV. A cell line was established from embryos of the insect C. chalcites. This cell line was shown to be permissive for both ChchNPV and the related Trichoplusia ni NPV (TnSNPV). This novel cell line will be a useful tool for making ChchNPV phr mutant viruses to study the impact of DNA repair mediated by photolyases on baculovirus ecology. The collected data support the hypothesis that the Cc- phr2 gene provides a baculovirus with an ecological benefit by increasing the resistance to UV.