Portrait of a Postdoc - Mei Yu
Baculoviridae represents a family of large rod-shaped enveloped viruses containing a large circular double stranded DNA genome. These viruses only replicate in invertebrates. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the genus Nucleopolyhedrovirus in the family Baculoviridae. AcMNPV DNA replication in Spodoptera frugiperda (Sf) cells occurs in the nuclei of infected cells, and is first detected 6 to 8 hours post-infection. Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) is a host specific virus for spruce budworm (Choristoneura fumiferana), so CfMNPV has potential for use in forestry as a biological pest control agent against the spruce budworm. Nine viral genes (ie-1, ie-2, p143, dnapol, lef-1, lef-2, lef-3, pe38 and p35) are involved in directing replication of plasmids carrying viral DNA inserts in transfected cells, suggesting that these genes are involved in baculovirus DNA replication. Our lab has demonstrated P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have also shown that LEF-3, a single-strand DNA binding protein, is required for transporting P143 into the nuclei of insect cells. The results of previous transient replication assays have suggested that the P143-LEF-3 complex may be a major host range factor for baculovirus replication.
Because the replication of CfMNPV and AcMNPV is host cell specific, I am investigating the possible role of the P143-LEF-3 complex in regulating species specificity of virus infection. The approach I am taking involves generating genetic knockouts (KO) of either P143 or LEF-3, or both genes in AcMNPV and replacing them with their CfMNPV homologues. The knockouts are being constructed in an AcMNPV artificial chromosome (bacmid), which replicates in both E. coli and Sf21 cells.
I have constructed linear DNA cassettes containing an antibiotic resistance gene flanked by 50 bp of sequence upstream and downstream of the appropriate AcMNPV gene specific open reading frames. These DNA cassettes are being electroporated into E. coli strains which express the lambda phage Red-mediated recombination genes and also carry the AcMNPV bacmid. I will be screening for clones which are antibiotic resistant, suggesting that either p143 or lef-3 has been replaced by the antibiotic resistant gene. I will then add back the CfMNPV homologues by insertion into the polyhedrin locus of the KO bacmids by using a donor plasmid carrying the CfMNPV genes and Tn7-based transposition. The donor plasmids carry the AcMNPV polyhedrin gene plus the CfMNPV p143 or lef-3 gene under the control of appropriate AcMNPV promoter. I will then investigate, by real-time PCR, the ability of the recombinant bacmids to replicate in either Sf or Cf cells. Rescue bacmids in which the AcMNPV p143 and lef-3 gene inserted into a donor plasmid will serve as positive controls. The final experiments in this project will replace both AcMNPV P143 and LEF-3 with their CfMNPV counterparts to determine whether P143 and LEF-3 complex plays a role in species specificity of virus infection in nonpermissive cells.
For further information, contact Dr. Carstens