Functional Study Of Autographa Californica Multiple Nucleopolyhedrovirus Odv-e18

Baculovirus host range is limited to Arthropods, mainly in the Lepidoptera, Hymenoptera and Diptera. Baculoviruses contain dsDNA genomes ranging from approximately80to approximately180kbp in size. The baculovirus Autographa californica multiple nucleopolyhedrovirus(AcMNPV) has a134kbp genome that has about150predicted ORFs. odv-e18(el8, ac143) is the143ORF of AcMNPV. The homologous genes of e18distribute in all baculovirus which had been sequencd. In this paper, we try to study the functions of e18in virus life cycle. The main results are as bellows:Knockout e18from AcMNPV genome through λ-Red homologous recombination. Replacing1-63bp sequence of the e18with the chloramphenicol acetyltransferase gene. Finally, we constructed e18knockout bacmid vAce18KO.1. e18knocked out (vAce18KO-gfp-pH), repaired (vAce18KO-Rep-gfp-pH) and wild type AcMNPV (vAcgfp-PH) viruses with gfp gene and polh gene were constructed through Bat-to-Bac system. After transfection-infectection assays, we found that e18was essential for BV production. Knockout of e18resulted that BV could not propagate, while polyhedron could be formed.2. Earlier expression mutants were constructed by using AcMNPV early gene iel promoter to control the expression of e18. We built two types mutant bacmid, such as vAcPiel-e18-gfp-PH and vAcwt-Piel-el8-gfp-PH We concluded that the first of earlier expression of e18had led to a defecting infectious BV production, without effecting on polyhedron formation. The other mutant bacmid has no effection on the production of BV and the formation of polyhedron.3. Overexpression mutants were constructed by using AcMNPV very late gene p10promoter to control e18expression. We built a mutant bacmid, vAce18KO-rep-Pp10-e18-PH. Through the transfection experiments, we have foud that this mutant made the number of cells that produce occlusion bodies reduced.4. E18protein was mainly distributed in the cytoplasm at12h p.i.; most of E18was remainly distributed in the cytoplasm at18h p.i., while only little of E18was in nucleus; all of E18was in nucleus at24h p.i., and during the late phase of infection E18 was located mainly to the ring zone.