Functional Study Of Autographa Californica Multiple Nucleopolyhedrovirus E25

Baculoviruses are arthropod-specific DNA viruses, with production of two virion phenotypes in their life cycles, the budded virus (BV) and the occlusion-derived virus (ODV). Expression of viral genes can be divided into early, late and very late according to the cascade regulation in transcription. Comparative analysis of all sequenced57baculovirus genomes to date shows that there are33identified genes in common, called core genes. e25is a late gene, identified as one of the core genes. The product is associated with both BV and ODV envelop, while function in virus replication is unknown yet.Here, we try to study the functions of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) e25gene in virus life cycle. The main results are as bellows:1. AcMNPV bacmid bMON14272was used to generate the e25knockout bacmid by homologous recombination in Escherichia coli using the λ Red system. A591bp sequence of e25from nt79,972to80,562was replaced by chloramphenicol resistance gene (cat). We constructed e25knockout bacmid vAce25ko successfully.2. e25knocked out (vAce25ko-PH-gfp), repaired (vAce25ko-rep-PH-gfp) and wild type AcMNPV (vAcPH-gfp) viruses with polh gene and gfp gene were constructed through Bac-to-Bac system. After transfection-infect assays and the analysis of virus growth curve, we found that e25was essential for BV production. Knockout of e25resulted that BV could not propagate, while polyhedron could formed.3. Earlier expression and overexpression mutants were constructed by using AcMNPV early gene iel and very late gene p10promoters to control e25expression. We concluded that earlier expression of e25led to a defect in infectious BV production, without effect on polyhedron formation. Overexpression of e25didn’t affect viral propagation.4. By monitoring gus expression under early gene dnapol or late gene vp39promoters, we found that neither knockout nor earlier expression had impact on early gene expression, knockout of e25didn’t affect late gene expression while earlier expression did inhibit the late gene expression a little.5. Nuclear actin polymerization was essential for viral replication. Since both E25and F-actin were found in viral storms, specific staining of F-actin with rhodamine phalloidin was performed to confirm whether they are associated. We observed that F-actin was formed in the nucleus of transfected cells, which suggests that knockout and earlier expression of e25didn’t affect F-actin formation induced by baculovirus infection.6. E25protein was mainly distributed in the cytoplasm at24hpt, then transported into nuclear periphery at36hpt, with small amount of distribution in cytoplasm. At48h and72hpt, E25was mainly concentrated in the nucleus. These implied that E25in the transfected cells was transported from cytoplasm to nucleus.7. Seven mutant plasmids were constructed by inserting egfp to the1st,45th,118th or227th codon of e25, or substituting2to45aa,46to117aa or118to227aa with egfp. Then we transposed the resultant plasmids into vAce25ko or bMON14272to obtain14bacmids. After transfection and infection assays, we found that only mutant with normal e25and e25substituting2to45aa with egfp could propagate normally, mutant with normal e25and e25substituting46to117aa could get a little propagation, mutant with egfp fused with e25at C terminal could replicate little, mutant with egfp fused with e25at N terminal couldn’t replicate totally. These results indicate that all mutants would affect virus propagation.8. By immunofluorescence assay, we found that in the condition of absence of normal e25, when2to45aa was substituted by egfp, or egfp was inserted before the2nd condon, E25could not enter into nucleus; mutant with egfp fused with e25at C-terminal has no impact on E25location.; all other mutants would result in defect of E25entrance into nucleus. The sublocalizations of E25in nucleus were affected as well. In condition of normal e25presence, when fused with egfp at C terminal, E25mutant has no impact on the localization of normal E25, all other mutants would inhibit the E25entrance into nucleus. These implied that N-terminal of E25was essential for its entrance into nucleus and E25probably functioned as a polymerization during virus life cycle.