Functional Analysis Of Iap-like Genes In Heliothis Virescens Ascovirus 3h (HvAV-3h)

Ascoviruses belong to a new family which is a subbranch of large insect viruses with circular dsDNA genomes.Ascoviruses cause a chronic and fatal disease when they attack Lepidopteran insect larvae,most of them belonge to Noctuidae.The cell initiates with the enlargement of nuclei in ascoviruses-infected cells and forms of virion-containing vesicles.Vesicles are formed,separated,and accumulated almost in the hemocoel making it milky-white.The process of vesicles formation is very similar to the formulation of apoptotic bodies.Rather than driving cell death,the developing apoptotic bodies was rescued by the synthetic viral protein to form vesicles.In this study,HvAV-3h was chosen as the target to provide a new theoretical basis for confirming the relationship between HvAV-3h and apoptosis and the genes played roles in apoptotic process induced by HvAV-3h.The main results are as follows:In the present study,a quick method for the determinating ascovirus stocks titer was developed based on ascovirus-induced apoptosis in infected insect cells.This method aimed to determine the optimal time of ascovirus-induced apoptosis in infected cells by flow cytometry and microscopic examination,and the optimal time of HvAV-3h expressed in IOZCAS-Spex-II-A cells is 24 h p.i..Cells infected with serial dilutions of virus(10^-2 to10^-10)for 24 h were stained with trypan blue.The stained cells were counted,and the percentage of nonviable cells was calculated.The stained cell rate was compared between virus-infected and control cells.The well of the minimum-dilution group that had a significant difference compared with control and the well of the maximum-dilution group that had no significant difference were marked as positive wells if the stained cell rate was higher than the average stained cell rate of control wells;otherwise,the wells were marked as negative wells.Subsequently,the virus titer calculated through the method of Reed-Muench was 5.62×10⁸ TCID₅₀/mL.The results showed that HvAV-3h was observed to cause apoptosis as early as 12 h p.i.in IOZCAS-Spex-II-A cells,however,HvAV-3h suppressed chemically induced apoptosis treated with ActD after 24 h p.i..The genome of HvAV-3h possessed five inhibitor of apoptosis genes?iap?,designated as iap-like gene-1?IAP-1?to iap-like gene-5?IAP-5?.Domain analysis showed that only IAP-2 contains an imperfect baculoviral IAP repeat?BIR?domain and a RING domain.Expression profile showed that iap-like gene-1and iap-like gene-2 had a transcript peak at 9 h p.i.,another peak at 21 h p.i.and 27 h p.i.,respectively.Iap-like gene-3 only had a expressed peak at 24 h p.i.,however,the eapression of iap-like gene-4 and iap-like gene-5 did not significantly change during HvAV-3h infection.Transient expression of IAPs showed that each of these IAPs alone did not have any pro-apoptosis function.Transient expression of iap-like genes and the stable transfectants treated with ActD revealed that only IAP-2 was a functional apoptosis inhibitor that suppressed apoptosis induced by ActD,while other four IAPs alone did not show any anti-apoptosis activity.These results indicated that IAP-2 might play a role in blocking apoptosis during HvAV-3h infection.