The microRNA pathway activation in insect cell model upon Autographa californica multiple nucleopolyhedrovirus infection

Background:The immune system of insects exerts fundamentally different antiviral mechanisms than mammals.MicroRNAs(miRNAs)play vital roles in developing insect antiviral immunity.MiRNAs expression profiles of insects changed significantly during baculovirus infection.Methods:Differential expression profiles of miRNAs in Spodoptera frugiperda were monitored by next-generation sequencing(NGS)and RT-qPCR during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.The transcription levels of genes were detected by RT-qPCR.The 50%tissue culture infective dose(TCID_(50))endpoint dilution assay was used to determine the proliferation of progeny virus.Results:NGS revealed that 49 miRNAs were differentially expressed in Sf9 cells,and 10 of them were significantly up-or down-regulated.Though RT-qPCR analysis,we observed the similar trends for the expression patterns of significantly differentially expressed miRNAs from NGS.Moreover,the transcription levels of core genes,Exportin5,Dicer1,and Argonaute1,in miRNA biogenesis pathways were significantly increased after AcMNPV infection.For five selected miRNAs,miR-34-5p could regulate the proliferation of baculovirus progeny virus and energy metabolism.Conclusion:The miRNAs biogenesis pathway in Sf9 cells plays an important role and may be stimulated to resist AcMNPV infection.This work provides evidence for the molecular mechanism of baculovirus-insect interaction and offers novel ideas and directions for green pest control technology.

Systematic Analysis of 42 Autographa Californica Multiple Nucleopolyhedrovirus Genes Identifies An Additional Six Genes Involved in the Production of Infectious Budded Virus

Baculoviruses have been widely used as a vector for expressing foreign genes.Among numerous baculoviruses,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is the most frequently used and it encodes 155 open reading frames(ORFs).Here,we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses(BVs)by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays.The results showed that among the 39 functionally unverified genes and 3 recently reported genes,36 are dispensable for infectious BV production,as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus.Three genes(ac62,ac82 and ac106/107)are essential for infectious BV production,as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus.In addition,three genes(ac13,ac51 and ac120)are important but not essential for infectious BV production,as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses.We then grouped the 155 AcMNPV genes into three categories(Dispensable,Essential,or Important for infectious BV production).Based on our results and previous publications,we constructed a schematic diagram of a potential mini-genome of AcMNPV,which contains only essential and important genes.The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.

Autographa Californica Multiple Nucleopolyhedrovirus orf13 Is Required for Efficient Nuclear Egress of Nucleocapsids

Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)orf13(ac13)is a conserved gene in all sequenced alphabaculoviruses.However,its function in the viral life cycle remains unknown.In this study,we found that ac13 was a late gene and that the encoded protein,bearing a putative nuclear localization signal motif,colocalized with the nuclear lamina.Deletion of ac13 did not affect viral genome replication,nucleocapsid assembly or occlusion body(OB)formation,but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus.Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm,while the OB morphogenesis was unaffected.Taken together,our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding,but was dispensable for OB formation.

Polyhedrosis Virus in Bacillus thuringiensis

This study was conducted to build a recombinant strain with highly insecticidal activity and a wide host range by using the crylAc and p74 gene. Firstly, the p74 gene was amplified from the genosome ofAutographa californica multicapsid nucleopolyhedrovirus. The crylAc gene and the terminator gene of crylAc, named crylAct, were amplified from the plasmid of Bt 4.0718 strain. Three T vectors, named pTp74, pT1Ac, and pT1Act which held the aimed gene p74, cry1Ac, and crylAct, respectively, and two middle vectors, named pTp74Act and pTIAcp74 which held the aimed fusion gene p74-crylAct and cry lAc-p74, respectively, were built by using pMDI 8-T. Then pTiAcp74 and the shuttle plasmid were digested and linked and an expressing-vector pH1Acp74 was built. Finally, pH1Acp74 was transformed into the acrystalliferous strain XBU001 and the aimed recombinant strain XBU-H1Acp74 was obtained. The expression of Bt transformant XBU-H1Acp74 was analyzed by SDS-PAGE which showed XBU-H1Acp74 could produce 130 kDa CrylAc protein and 50 kDa P74 protein. The insecticidal activity of transformant against Spodoptera exigua was evaluated compared with the contrast strains HTX-42 （only crylAc gene was transformed into XBU001） after autolysis. The LCs0 of HTX-42 was higher than that of the XBU-H IAcp74＇s, which implied that P74 could increase the efficacy and range of Bt Cry toxins in insect control. The fusion gene of crylAc and p74 were constructed successfully which will be served as the foundation lbr constructing the fusion genes of Bt cry gene and other foreign genes.

The role of viral protein Ac34 in nuclear relocation of subunits of the actin-related protein 2/3 complex

The actin nucleator actin-related protein complex(Arp2/3) is composed of seven subunits: Arp2,Arp3, p40/ARPC1(P40), p34/ARPC2(P34), p21/ARPC3(P21), p20/ARPC4(P20), and p16/ARPC5(P16). Arp2/3 plays crucial roles in a variety of cellular activities through regulation of actin polymerization. Autographa californica multiple nucleopolyhedrovirus(Ac MNPV), one of the beststudied alphabaculoviruses, induces Arp2/3 nuclear relocation and mediates nuclear actin polymerization to assist in virus replication. We have demonstrated that Ac34, a viral late-gene product, induces translocation of the P40 subunit of Arp2/3 to the nucleus during Ac MNPV infection. However, it remains unknown whether Ac34 could relocate other Arp2/3 subunits to the nucleus. In this study, the effects of the viral protein Ac34 on the distribution of these subunits were studied by an immunofluorescence assay. Arp2, P34, P21, and P20 cloned from Spodoptera frugiperda(Sf9) cells showed mainly cytoplasmic localization and were relocated to the nucleus in the presence of Ac34. In addition, Arp3 was localized in the cytoplasm in both the presence and absence of Ac34, and P16 showed whole-cell localization. In contrast to Sf9 cells, all subunits of mammalian Arp2/3 showed no nuclear relocation in the presence of Ac34. Co-immunoprecipitation analysis of the interaction between Ac34 and Arp2/3 subunits revealed that Ac34 bound to P40,P34, and P20 of Sf9 cells. However, none of the subunits of mammalian Arp2/3 interacted with Ac34, indicating that protein-protein interaction is essential for Ac34 to relocate Arp2/3 subunits to the nucleus.

Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome

Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides.Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome.Recently, we generated a synthetic baculovirus Ac MNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV). Here, we report the modification of Ac MNPV-WIV-Syn1 into a novel bacmid, Ac Bac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a Lac Z:att Tn7 and egfp cassette was constructed, and recombined with a linearized Ac MNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid Ac Bac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the att Tn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that Ac Bac-Syn can be readily used for foreign gene insertion and expression.Ac Bac-Syn has several advantages over the conventional Ac MNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration;its DNA copy numbers could be induced to a higher level in E. coli;and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.