Studies On The Structure, Function And Potential Killing Activity Of P13 In Group Ⅱ Nucleopolyhedrovirus

p13 gene of Leucania separata multinuclear polyhedrosis virus (Ls-p13) was first described by our laboratory as early as 1995. With the large-scale genome sequencing, more than 20 p13 homologues in baculoviruses have been reported. Interestingly, all the reported p13 genes were specifically present in Group II nucleopolyhedroviruses (NPVs) and granuloviruses (GVs), but not in Group I NPVs. The character of p13 promoter and the possible functions of P13 have been predicted in several articles, but there is no evidence in experiment by far. In this study, we have studied on p13 promoter activity, its phylogenetic tree, its killing activity in homologous and heterologous system and possible mechanism. The work set up the foundation for further study of P13 function and its potential application as an opponent of bio-insecticide.Twenty-two p13 sequences from fourteen kind of baculoviruses were reported to date. Fourteen sequences ( each represents one kind of baculovirus P13) selected from the above bacouloviruses genome and were analyzed by DNASTAR software. All the p13 genes share homology from 42.1%-74.7% in amino acid. Among all the sequences, Ls-p13 share the highest homology with HaSNPV p13 in amino acid (58.7%), while share the lowest homology with PoGV p13 in amino acid (44%). Based on the phylogenetic relationship of amino acid, P13 can be clearly divided into two groups in phylogeny, which is p13 genes of GVs and Group II NPVs, respectively. p13 genes of GVs are closely related (58.2%-74.7% amino acid identity) while those of Group II NPVs appear to be more divergent (44.4%-73% amino acid identity). It can be concluded that GV may form a single group according to their close relationship of p13 gene, while NPV containing p13 gene could be divided into subgroup I (LsNPV,HaNPV and SlNPV) and subgroup II (other group II NPVs).p13 gene contains both GTGTTATA box and CAT/AT box of the early promoter, a TTAAG box of late promoter in its 5'-UTR, a mini cistron following the core sequence of the early promoter, and homologue repeats (hr) which is universe in baculovirus. Generally, there are many hrs in baculovirus and the number of p13 hr is different among different baculoviruses. It has been proved that hrs in baculovirus is the ori of DNA replication and enchancer of transcription. hr at p13 gene 5'-UTR is also important for early gene high-expression. Our work confirmed that p13 transcription was regulated by both early and late promoter. Interestingly, we found that the hr4 enhancer/Ha-p13 promoter not only has no host specificity, but also increases its activity in heterogeneous Sf9 cells nearly 100 times more than in homologous Hz-AM1 cells. We presume that some viral or cellular factors may control the normal low-expression of P13 in host cells.In order to make the work more representative, we choose Ha-P13 from Heliothis armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) and Ls-P13 from LsNPV to study the possible killing activity in homologous and heterologous system, respectively. As Ha-p13 was present in the genome of HaSNPV-G (G represents eGFP) and G was not fused with any viral genes, dsRNA-Hap13 could be used as RNA interference to study the effect of Ha-P13 on HaSNPV-G infection. Our results demonstrated that dsRNA-Hap13 could specifically knockdown Ha-P13 expression in both homologous Hz-AM1 cells and heterologous Sf9 cells. Co-injection 5ug dsRNA-Hap13 with HaSNPV-G in larvae could decrease the killing rate and the efficacy was dsRNA-Hap13 dose dependent. Thus p13 gene was indicated as a killing associated gene. On the other hand, as p13 gene was absent in group I NPVs such example as baculovirus AcMNPV, we inserted Ls-p13 gene into AcMNPV to evaluate its killing activity in heterologous system. Bioassay results clearly showed that Ls-P13 could accelerate the killing rate of AcMNPV, but the effect is abolished when its LZLD is disrupted. Such effects were verified both when the protein Lsp13 was expressed in vivo and when a purified sample of the protein was injected into the larvae. Although the mechanism of P13 is still unknown, its killing property is attractive and will promote the further study of P13 functions and its potential applications in biopesticides.In order to probe the possible mechanism of Ls-P13's killing activity in heterologous system, we constructed a series of recombinant AcMNPVs to study Ls-P13 efffect on polyhedra and BV yield. Phase-contrast microscope and TEM results indicated that Ls-P13 could decrease the polyhedra yield and the inhibition rate decreased with Ls-P13 expression from early to late phase, while the efficacy was lost when LZLD of Ls-P13 was mutated. However, when both transmenbrane domains were deleted, Ls-P13 suppression on polyhedra was further enhanced. On the other hand, our results indicated that BV increased nearly three times when Ls-P13 was highly expressed at early phase. As long as LZLD was mutated, Ls-P13's effect on BV yield was also lost. However, its increase on BV was still slightly accelerated after the TM deletions. From above results, we concluded that Ls-P13 decreased the yield of polyhedra while increased the yield of BV. LZLD was one of important regions for this efficacy. TMs affected this efficacy only by changes of Ls-P13 distribution. The lost of balance between polyhedra and BV yield may be one reason of Ls-P13 killing activity in heterologous system.Moreover, we have studied the location of P13 in homologous Hz-AM1 cells and heterologous Sf9 cells. Confocal results showed that P13 was located in the cytoplasm membrane at 48h no matter in homologous Hz-AM1 cells or heterologous Sf9 cells. Ls-P13 localization in heterogeneous Sf9 cells was a dynamic process: most of Ls-P13 distributed in the nuclei at early stage (12h); Ls-P13 was then (24h) transferred from nuclei to cytoplasm; Ls-P13 was finally localized cytoplasm membrane. Ls-P13 distribution was transferred from cytoplasm membrane to nuclei with the deletion of TMs, especially after both TM1 and TM2 deletions. Polyhedra are produced in the nucleus while BVs are budded at the cytoplasm membrane. As Ls-P13 distribution was decreased in cytoplasm membrane but increased in nucleus with TM domains deletion, it is well explained why the effect of Ls-P13 with TM mutation on BV increase became weaker while the repression on polyhedra yield was stronger than that of wide type Ls-P13. On the other hand, Ls-P13 would be early-high expressed and enriched in nucleus when Ls-P13 was droved by hr5/IE1 promoter. However, Ls-P13 would move from nucleus to cytoplasm membrane with its expression phase being postponed. It is also explained why Ls-P13 repression efficacy on polyhredra was decreased with its expression from early phase to late phase.