Helicoverpa armaigera nucleopolyhedrovirus ORF50 is an early gene not essential for virus propagation in vitro and in vivo

Homologs of Helicoverpa armigera nucleopolyhedrovirus ORF50 (HA50) are found in most alphabaculoviruses, but their functions remain unknown. Here, we characterized whether Ha50 is indispensable for virus progration. Ha50 transcript was first detected at 3 h post-infection from HearNPV-infected HzAM1 cells. 3′RACE analysis showed that Ha50 transcript was polyadenlylated. 5′RACE analysis revealed two transcription initiation sites, one of which was mapped to the canonical baculovirus early transcription initiator motif CAGT. HA50 protein could be detected from infected cells harvested at 12 h post-infection. Transient expression assays showed that GFP-fused HA50 localized in the cytoplasm and nucleus of HzAM1 cells with or without superinfection. To further examine the role of Ha50 in the virus life cycle, a Ha50 knockout bacmid and a repair bacmid carrying Ha50 under the control of its native promoter elements were constructed using bacmid technology. One-step growth curve analysis showed that the kinetics of infectious budded virus production of Ha50 knockout virus was similar to that of the parental virus or the repair virus. Analysis of the expression of viral early protein IE-1, late protein VP39 and very late protein suggested that viral protein expression was not affected by Ha50 inactivation. Electron microscopy revealed that HaBacΔ50-PH-G occluded viruses (ODVs) and occlusion bodies were indistinguishable from those of the wild-type virus. Similarly, bioassays showed no significant difference in the LC50 values between Ha50 deletion virus and wild-type virus. Our results together demonstrate that Ha50 is an early gene dispensable for virus propagation in vitro and in vivo.