Roles Of Host Endocytic Pathway Related Factors In Infection Of Autographa Californica Multiple Nucleopolyhedrovirus

Baculoviruses is a group of large,double-stranded DNA enveloped viruese.In a typical life cycle,baculovirues usually produce two kinds of virions with distinct morphology,budded virions(BV)and occlusion-derived virions(ODV).ODV targets the midgut epithelial cells of insect hosts and initiate the primary infection,whereas BV infects the other tissue cells or cultured cell lines in vitro.Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is the type species of baculoviruses and has been extensively studied.The budded virions of AcMNPV enter cells via calthrin-mediated endocytosis.Transcriptome analysis revealed that,in AcMNPV BV infected Trichoplusia ni Tnms42 cells,the transcription levels of dynamin,rab5,and rabll,which serve as the critical regulator of cellular endocytosis,were significantly up-regulated.However,the functional role of these cellular factors in AcMNPV BV infection is not clear.In this study,to confirm the transcriptome data,we initially detected the transcript level of dynamin and four rab genes(rab4,rab5,rab7,rabll),which function as the dominant regulator of early,late or recycling endosomes,in AcMNPV-infected Spodoptera frugiperda Sf9 cells.Our results showed that similar with that of transcriptome analysis,at the early stage of infection(0-6 h),the transcript levels of dynamin,rab5 and rabll are significantly up-regulated.Using dynasore,a specific inhibitor of dynamin,to treat Sf9 cells or transfection Sf9 cells with double-strand RNA to down-regulate the transcription of dynamin dramatically decrease infectious BV production.Further analysis revealed that,dynasore treatment did not affect BV binding of Sf9 cells but significantly reduced the entry efficiency of budded virions.Confocal microscopy analysis showed that the internalized mCherry-labeled viruses are colocalized with GFP-tagged Rab5 and Rabll.Overexpression of the dominant-negative(DN)forms of Rab5 and Rabll or RNA interference down-regulation of the expression the expression of rab5 and rabll significantly reduced the infectious BV production.Further analysis showed that overexpression of DN forms of Rab5 and Rabll has no effect on BV binding of Sf9 cells but dramatically decreased the entry efficiency of budded virions.In contrast,overexpression of the DN forms of Rab7 or Rabll or RNAi down-regulation of the expression of rab7 or rabll has no effect on infectious BV production.Direct release of nucleocapsids into cells expressing DN forms of Rab5 or Rabll by low-pH triggered fusion between viral envelopes and cellular membranes or transfection Sf9 cells with recombinant bacmid DNA expressing DN forms of Rab5 or Rabll did not affect the infectious BV production,suggesting the requirement of Rab5 and Rab11 for efficient entry but not for budding and release of progeny budded virions.Additionally,RNAi down-regulation of the expression of the components of the endosomal sorting complex required for transport-Ⅲ(ESCRT-Ⅲ),such as Vps2B,Vps20,Vps24,Snf 7,Vps46,or Vps60 dramatically decreased the efficiency of entry and egress of BV.our data validate the model in which AcMNPV BV enter permissive host cells by clathrin-coated vesicles and de-envelopment of BV to release the nucleocapsid occurs predominantly within early endosomes and maturing endosomes rather than within late endosomes.Additionally,the results from Rab11 suppression studies support a model in which recycling endosomes may be involved in recycling some of the entering AcMNPV BV to the plasma membrane and this strategy may rapidly spread virions to neighbor cells to establish a productive infection.