Our long-term goal is to learn to control (expand and restrict) the host range of baculoviruses. In this project our aim was to expand the host range of the prototype baculovirus Autographa cali/arnica nuclear polyhedrosis virus (AcMNPV) towards American and Israeli pests. To achieve this objective we studied AcMNPV infection in the non-permissive hosts L. dispar and s. littoralis (Ld652Y and SL2 cells, respectively) as a model system and the major barriers to viral replication. We isolated recombinant baculoviruses with expanded infectivity towards L. dispar and S. littoralis and tested their infectivity towards other Lepidopteran pests. The restricted host range displayed by baculoviruses constitutes an obstacle to their further implementation in the control of diverse Lepidopteran pests, increasing the development costs. Our work points out that cellular defenses are major role blocks to AcMNPV replication in non- and semi-permissive hosts. Therefore a major determinant ofbaculovirus host range is the ability of the virus to effectively counter cellular defenses of host cells. This is exemplified by our findings showing tliat expressing the viral gene Ldhrf-l overcomes global translation arrest in AcMNPV -infected Ld652Y cells. Our data suggests that Ld652Y cells have two anti-viral defense pathways, because they are subject to global translation arrest when infected with AcMNPV carrying a baculovirus apoptotic suppressor (e.g., wild type AcMNPV carryingp35, or recombinant AcMNPV carrying Opiap, Cpiap. or p49 genes) but apoptose when infected with AcMNPV-Iacking a functional apoptotic suppressor. We have yet to elucidate how hrf-l precludes the translation arrest mechanism(s) in AcMNPV-infected Ld652Y cells. Ribosomal profiles of AcMNPV infected Ld652Y cells suggested that translation initiation is a major control point, but we were unable to rule-out a contribution from a block in translation elongation. Phosphorylation of eIF-2a did not appear to playa role in AcMNPV -induced translation arrest. Mutagenesis studies ofhrf-l suggest that a highly acidic domain plays a role in precluding translation arrest. Our findings indicate that translation arrest may be linked to apoptosis either through common sensors of virus infection or as a consequence of late events in the virus life-cycle that occur only if apoptosis is suppressed. ~ AcMNPV replicates poorly in SL2 cells and induces apoptosis. Our studies in AcMNPV - infected SL2ceils led us to conclude that the steady-state levels of lEI (product of the iel gene, major AcMNPV -transactivator and multifunctional protein) relative to those of the immediate early viral protein lEO, playa critical role in regulating the viral infection. By increasing the IEl\IEO ratio we achieved AcMNPV replication in S. littoralis and we were able to isolate recombinant AcMNPV s that replicated efficiently in S. lifforalis cells and larvae. Our data that indicated that AcMNPV - infection may be regulated by an interaction between IE 1 and lED (of previously unknown function). Indeed, we showed that IE 1 associates with lED by using protein "pull down" and immunoprecipitation approaches High steady state levels of "functional" IE 1 resulted in increased expression of the apoptosis suppressor p35 facilitating AcMNPV -replication in SL2 cells. Finally, we determined that lED accelerates the viral infection in AcMNPV -permissive cells. Our results show that expressing viral genes that are able to overcome the insect-pest defense system enable to expand baculovirus host range. Scientifically, this project highlights the need to further study the anti-viral defenses of invertebrates not only to maximi~e the possibilities for manipulating baculovirus genomes, but to better understand the evolutionary underpinnings of the immune systems of vertebrates towards virus infection.
Inhibition of retroviral replication by anti-sense RNA.
