폴리드나바이러스(polydnavirus: PDV)는 일부 내부기생봉에 공생하는 이중나선형 DNA 바이러스 분류군이다. Cotesia plutellae bracovirus (CpBV)는 프루텔고치벌(C. plutellae)에 공생하는 일종의 PDV이다. 프루텔고치벌은 어린 배추좀나방(Plutella xylostella) 유충에 기 생한다. 기생 초기에 발현하는 CpBV-ELP1 유전자는 혈구세포에 세포독성을 발휘하면서 기주의 세포성 면역을 억제하여 기생에 중요한 역할을 담당하고 있다. 본 연구는 이 유전자를 담배 식물에서 발현하여 해충에 대한 경구독성을 분석하는 데 목적을 두었다. 재조합 CpBV-ELP1 단백질 이 배큘로바이러스 발현시스템을 통해 합성되어 세포배양액에 분비되었다. 수거된 세포배양액은 일련의 단백질 분리과정(ammonium sulfate 단백질 분획, size exclusion 크로마토그래피, 이온교환 크로마토그래피)을 통해 CpBV-ELP1 단백질을 분리하는 데 이용되었다. 분리된 rCpBV-ELP1 단백질은 파밤나방(Spodoptera exigua) 혈구에 대한 뚜렷한 세포독성을 보였다. CpBV-ELP1은 파밤나방 5령충에 대해서 혈강 주입하여 살충력을 나타냈고, 엽침지법을 이용하여 경구독성을 갖고 있는 것을 확인하였다. CpBV-ELP1 유전자를 CaMV 35S 유전자 프로모터 와 opaline synthase 유전자 전사종결신호를 갖는 pBI121 벡터에 클로닝하여 Agrobacterium tumefaciens LBA4404 세균에 형질전환을 유도하였 다. 형질전환된 세균은 담배(Nicotiana tabacum Xanthi)잎에 감염하여 캘러스를 유도하게 하였고 이후 차세대(T1)를 확보하였다. T1 세대 담배 는 파밤나방에 대한 해충저항성을 갖고 있음을 확인하였다. 이러한 결과는 CpBV-ELP1 유전자가 형질전환작물을 통해 해충방제에 응용될 수 있 다는 것을 제시하고 있다.

Polydnavirus (PDV) is a group of double-stranded DNA insect viruses. PDV is mutualistic with some ichneumonid and braconid wasps to parasitize specific lepidopteran hosts. The viral genome is located on the wasp chromosome(s) as a proviral form and replicates only at the female reproductive organ during late pupal stage. The viral particles are accumulated in the oviduct lumen and delivered to the parasitized host along with wasp eggs during parasitization. The viral particles enter target tissues in the parasitized larvae and alter host physiological processes for the wasp development by suppressing immune responses and extending larval period. Cotesia plutellae bracovirus (CpBV) is a PDV symbiotic to C. plutellae parasitizing young larvae of the diamondback moth, Plutella xylostella. The viral particles of CpBV encode 157 open reading frames classified into different gene families. CpBV-PTP family is the largest and comprises of at least 40 gene members. CpBV-BEN, CpBV-ELP, and CpBV-IkB families also share common motifs in each gene group. In addition, two homologous genes of CpBV15α and CpBV15β are encoded in a viral genome segment. To apply these viral genes to enhance an alpha-baculovirus in insecticidal activity, they were recombined with AcNPV under a PDV promoter. As a control, under the same promoter, the recombinant baculovirus expressed an enhanced green fluorescence protein (EGFP). Upon injection or oral feeding tests, three different recombinant baculoviruses (AcNPV-ELP1, AcNPV-CpBV15α, AcNPPV-CpBV15β) enhanced the insecticidal activity compared to a control recombinant (AcNPV-EGFP). However, there was a variation in the up-regulation of the insecticidal activities among the recombinants. AcNPV-ELP1 showed the greatest potency in the insecticidal activity against the beet armyworm, Spodoptera exigua, larvae. AcNPV-ELP1 exhibited a significant variation in insecticidal activity among different larval stages of S. exigua. In the fifth instar, 1.435x107 PIB treatment of AcNPV-ELP1 showed a median lethal time at 112.7 h. ELP1 protein was detected in the hemolymph at 24 h after the viral treatment. Foliar spray of AcNPV-ELP1 was performed in pot assay and resulted in 88% control efficacy against S. exigua, while control efficacies of AcNPV-EGFP and bifenthrin (a pyrethroid insecticide) resulted in 65% and 96%, respectively. These results suggest that a PDV gene, ELP1, may be applied to develop a novel control agent by ameliorating commercial microbial insecticides or by generating transgenic crops.

Baculovirus expression system has been used to produce functional proteins of various eukaryotic genes. A polydnaviral gene, CpBV-ELP1, was cloned in an alpha-baculovirus, Autographa californica multiple nuclear polyhedral virus (AcNPV), and expressed in Sf9 cells. CpBV-ELP1 protein was released into the culture medium due to its signal peptide. The culture broth containing CpBV-ELP1 was collected and fractionated with different concentrations of ammonium sulfate. Most CpBV-ELP1 was precipitated in 25-100% ammonium sulfate. The precipitate proteins were separated with a size exclusion chromatography sieving 100 kDa size. CpBV-ELP1 was eluted after relatively high molecular weight protein peaks. The fractions rich in CpBV-ELP1 were collected and further fractionated with an anion exchange chromatography. The purified CpBV-ELP1 was toxic to both larvae of Plutella xylostella and Spodoptera exigua by oral test used as leaf dipping method. A lethal median concentrations (LC50) were 7.5 ㎍/mL (95% CI: 1.2-24.3 ㎍/mL) for 2nd instar larvae of P. xylostella and 4.4 ㎍/mL (95% CI: 1.9-8.4 ㎍/mL) for 3rd instar larvae of S. exigua. These results suggest that CpBV-ELP1 may be applied to develop novel transgenic crops.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), is a symbiotic provirus to an endoparasitoid wasp, C. plutellae. When the wasp parasitizes its natural host, Plutella xylostella, larvae, CpBV viral particles are translocated to hemocoel of P. xylostella along with the wasp eggs. CpBV-ELP1 is encoded in a viral segment and expressed in the parasitized larvae during entire parasitization period. A recombinant baculovirus expressing CpBV-ELP1 was constructed and applied to a non-natural host, Spodoptera exigua, larvae. When the recombinant baculovirus was injected to hemocoel, CpBV-ELP1 was expressed in hemocytes as early as 2h postinjection and then later expressed in other tissues. When it was applied to diet, CpBV-ELP1 was expressed in midgut epithelium at 12 h and subsequently expressed in internal tissues. Both application methods of the recombinant baculovirus caused significantly higher mortality of S. exiguathan non-recombinant baculovirus. Interestingly, midgut epithelial cells expressing CpBV-ELP1 by infection of the recombinant baculovirus showed poor cell-cell interactions. Integrin, a cell surface molecule associated with cell-cell interaction, was cloned in S. exigua and was confirmed in its expression in the midgut epithelium. A hypothesis was raised that CpBV-ELP1 interrupts integrin function by direct binding or by blocking internal integrin signaling.