Functional Analysis Of Orf107, Orf135 And Pkip Gene Of Helicoverpa Armigera Nucleopolyhedrovirus

Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is an important virusthat has been widely used in pest control in China. After genome sequence of theHearNPV, we are now focusing on elucidation of different genes of the virus. Inthis thesis, three of the ORFs, Hal07, Ha135 and Ha130 (pkip) were analyzed fortheir transcription, expression and localization. Their functions were studied bymaking knockout recombinant viruses.Ha107 of HearNPV encodes a putative protein of 51 kDa with homologs in afew groupâ…¡NPVs and a GV. Functional analysis of this ORF is described inChapter 2. Ha107 was transcribed in infected HzAM1 insect cells aspolyadenylated transcripts initiated at two distinct locations. By Western blotanalysis two forms of the HA107 protein were detected in HearNPV infected cells,a major form of 48 kDa and a minor form of 51 kDa. The HA107 protein was foundin the nucleocapsid fraction of both budded virions (BVs) and occlusion-derivedvirions (ODVs). BV infectivity of HaBacâ–³Ha107-eGFP-PH (Ha107 knockout virus)was much higher than that of 'control' virus and a Ha107-repair virus. Electronmicroscope analysis showed that the formation of occlusion bodies was notaffected by the deletion of Ha107. Our data indicate that Ha107 encodes anon-essential structural protein of HearNPV and that the deletion of this geneimproves the BV infectivity. transcribed as a polyadenylated transcript from 12 h post-infection in infected hostcells. Western blot analysis demonstrated the Ha135 encodes a 23 kDa protein,which was not present in budded virions (BV) or occlusion derived virions (ODV).When fused with green fluorescence protein, HA135 protein is predominantlydetected in the nuclei in host cells, with distinctive aggregates. Gel filtrationanalysis demonstrated that HA135 protein forms predominantly dimmer in vitro.Knockout Ha135 had no significant influence on polyhedra formation, BV growthcurve and ODV infectivity.The protein kinase interacting protein (pkip) gene is a consensus gene amongnucleopolyhedrovirus. In Chapter 4, the function of HearNPV pkip wasinvestigated. 3'RACE analysis demonstrated that HearNPV pkip was transcriptedas polyadenylated transcripts in HzAM1 cells from 8 h post infection. By Westernblotting, the product of HearNPV PKIP was found as a 20 kDa protein in infectedcells, close to the theoretical molecular weight. Upon viral infection, PKIP-GFPfusion protein was observed primarily to be located in nucleus. Deletion ofHearNPV pkip gene showed no impair to budded virus production and occlussionbody formation. Thus, we conclude that the pkip gene is not essential forHearNPV infection.