Studies On Apoptosis Induced By Spodoptera Litura Nucleopolyhedrovirus

The insect cell apoptosis induced by baculovirus infection limit the host range and wide application as bio-pesticide of baculovirus. Baculoviruses were able to block cell apoptosis or continue replication in apoptosis cells by long evolution. Most of baculovirus studied so far contain two or more anti-apoptosis genes. All of anti-apoptosis genes have not an ability to suppress apoptosis by studying in vitro. It is unclear that what function on earth are those anti-apoptosis genes not inhibiting the cell apoptosis in vitro. In this paper, the functions of Splt-iap4 and Splt-p49 of SpltNPV were studied in SpLi-221 cells by RNAi and in Sf9 cells by transient expression, respectively. Furthermore, the relationship between anti-apoptosis genes and their host evolution was discussed.A homology search of public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4); but their functions had not been investigated in its native host cells.In present study, RNA interference (RNAi) was used to silence the expression of Splt-iap4 and Splt-p49, respectively or synchronously, and to determine their roles during the SpltNPV life cycle. RT-PCR analysis and western blot analysis showed that the target gene expression had been knockdown in the SpltNPV-infected SpLi-221 cells after treating with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all of the cells had undergone apoptosis by 48 h. In contrast, viral replication progressed normally in infected cells treated with Splt-iap4 dsRNA. Interestingly, after the treatment with Splt-iap4 dsRNA, cells didn't congregate as those infected with SpltNPV in early infection phase, implying an unknown role of baculovirus iap. Transient expression of Splt-p49 could inhibit apoptosis in Sf9 cells induced by p35 mutant AcMNPV, but Splt-IAP4 couldn't. Collectively, our results show that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection.The antiapoptotic genes present in forty-three sequenced baculovirus genomes are summarized in table 5-2. Overall, different groups have their own different features: all lepidopteran-specific group I NPVs contain iap1 and iap2; all lepidopteran-specific group II NPVs but SpltNPV contain iap2 and iap3; iap5 are lepidopteran-specific GVs genes; hymenopteran-specific NPVs contain only iap3. That the high conservation of the different antiapoptotic gene types present in the different taxonomic groups implies apoptosis plays an important role in baculovirus phylogenesis.Spodoptera litura and Spodoptera exigua are both extremely serious agricultural lepidopteran pests and belong to the same family (Noctuidae) and the same genera (Spodoptera). Spodoptera litura nucleopolyhedrovirus (SpltNPV) and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) are viral pathogens to the two insects, respectively. Comparative analysis of the complete genome sequences of SpltNPV and SeMNPV showed two genomes are very similar. Although both the pathogens and the hosts are closely related, the mechanism of the two baculovirus not infecting the hosts each other is unclear. This study concentrated on investigating the pathology of SpltNPV-infected Se301 cells and the reason of the abortion infection.There were some pathology changes, for example, apoptosis bodies, congregation and vacuoles were observed on Se301 cells infected by SpltNPV from 24 h to 120 h post infection. Finally, it was not observed ODV (occlusion derived virus) on Se301 cells. Electro microscope and DAPI staining showed that the infected cells were the pathology of early apoptosis. The replication of virus had completed in SpltNPV-infected Se301 by testing with Dot blotting but finally, they did not produce infectious BV (budded virus). The results of RT-PCR analysis displayed that all genes from very early genes to late genes transcribed in the infected cells. Western blot analysis revealed that it was no specific Polyhedrin with a size of 29.7 kDa when using the Polyhedrin antiserum. Collectively, these results demonstrated that the SpltNPV-infected Se301 cells had obvious pathologic phenomenon that was deduced as early apoptosis and had not processed to the late phase namely apoptosis bodies. There were no infectious BV and ODV in the Se301 cells infected by SpltNPV, but the replication of virus and the transcription of all late genes had happened, although it was not detected the expression of very late genes, suggesting that early apoptosis is still one of the limited factors in viral replication cycle.