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.

Parasitism by an endoparasitoid wasp, Cotesia plutellae, results in significant immunosuppression of the diamondback moth, Plutella xylostella. Parasitized larvae significantly suffered higher susceptibility to a microbial biopesticide, Bacillus thuringiensis (Bt) than nonparasitized (NP) larvae. To find out an immunosuppressive agent causing the enhanced Bt efficacy, viral ankyrin (=vankyrin) genes encoded in C. plutellae bracovirus (CpBV) were analyzed by transient expression in NP larvae. CpBV segments containing different vankyrins were microinjected to NP larvae and expressed their encoded vankyrins. Expression of some vankyrins significantly inhibited immune response and enhanced Bt efficacy. This study suggests that expression of vankyrins suppress a cellular immune response and lose Bt tolerance of P. xylostella larvae.

An endoparasitoid wasp, Cotesia plutellae, contains a polydnavirus called C. plutellae bracovirus (CpBV) and induces various physiological alterations of parasitized host along with expressions of viral genes. Two host translation inhibitory factors (HTIFs) encoded in CpBV specifically inhibit host mRNAs at post-transcriptional level. They are expressed in late larval stage of Plutella xylostella parasitized by C. plutellae. To understand their late expression control, promoter region of an HTIF gene called CpBV15α was cloned by inverse PCR. The cloned HTIF upstream region (1,113 bp) possessed a putative JH response element (JHRE) and other promoter elements. The putative promoter region was rejoined with an open reading frame of enhanced green fluorescence protein (EGFP). When the recombinant vector construct was injected into early third instar larvae of nonparasitized P. xylostella, it was expressed in fourth larval instar at 72 h after injection, compared to relatively early expression in 24 h after injection of control construct containing a baculovirus immediate-early promoter. However, recombinant EGFP construct lost the late expression pattern when its promoter region was incomplete by truncating JHRE region. PYR application inhibited EGFP expression of the recombinant construct, but gave little influence on truncated constructs. Interestingly, when the complete promoter construct was injected to pupal stage, its late expression pattern was lost and showed early expression pattern. However, an addition of PYR to pupae, which had been injected with the complete promoter construct, inhibited the reporter gene expression. These results suggest that late expression of a HTIF (CpBV15α) is controlled by its promoter, which is sensitive to host JH titer.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), is symbiotic to an endoparasitoid wasp, C.plutellae, which specifically parasitizes young larvae of the diamondback moth, Plutella xylostella. A recent study on CpBV replication by analysis of ovary transcriptome of C.Plutellae suggests several candidate coat protein genes. This study was conducted to confirm the coat protein genes by analyzing coat proteins of CpBV viral particles by a tandem mass MALDI-TOF. Immunoprecipitation of ovary protein extract with a polyclonal CpBV antibody captured three proteins named as p35, p60, and p70. More number of coat proteins were resolved in a protein extract directly from viral particles. All candidate coat proteins are analyzed in amino acid sequences by MALDI-TOF. A comprehensive analysis of viral proteomics and ovary transcriptome determined novel viral coat proteins from CpBV

Parasitization by an endoparasitoid wasp, Cotesia plutellae, extends a larval period of Plutella xylostella and inhibits a larva-to-pupa metamorphosis. To determine antimetamorphic parasitic factor(s) in this host-parasitoid interaction, an effect of its symbiotic polydnavirus, Cotesia plutellae bracovirus (CpBV), was investigated by injecting purified virus particles to nonparasitized larvae of P. xylostella. Larvae injected with CpBV exhibited antimetamophosis in a viral dose-dependent manner. Also, the susceptibility to the viral injection was increased at young larval stages. Parasitized or virus-injected larvae shwed significant decrease in cell size of prothoracic gland and reduction in expression of ecdysone receptor (EcR) gene. However, they increased and maintained expression of insulin receptor (InR) gene. Twenty four CpBVsegments were individually injected to nonparasitized larvae. Only two segments (S22 and S27) had significant antimetamorphic effect. Subsequent RNA interference using double stranded RNA (dsRNA) was performed in each of encoded genes in each segment. Protein tyrosine phosphatase, ELP, and three hypothetical genes were determined to be antimetamorphic factors.

Polydnaviruses (PDVs) are a group of insect double stranded DNA viruses and symbiotically associated with host endoparasitoid wasps. Their segmented genome is located in host chromosome(s) in a proviral form. Viral replication is initiated at the ovary during late pupal stages. Little is known about the factors involved in the viral replication. This study analyzed the ovarian transcripts of an endoparasitoid wasp, Cotesia plutellae, by 454 pyrosequencing and subsequent gene annotation. Out of 2,226 contigs and 12,457 singletons, 50 transcripts categorized in DNA replication, coat proteins, and viral origins were selected as putative viral replication factors. The selected genes were analyzed in their expressions according to host wasp development. Quantitative real-time RT-PCRs showed that some of the selected genes were expressed during the viral replication at late pupal stage. Using RNA interference, five putative genes were tested in their implication in the viral replication by analyzing viral DNA amplification, structure of ovarian calyx, and parasitism. RNA interference of contig#1004 (broad complex) or contig#174 (a viral DNA polymerase gene) significantly inhibited DNA amplification without any impairment of viral formation, and subsequently resulted in significant reduction in the wasp parasitism. This study reports that two wasp genes (or not encapsidated viral genes) are implicated in the viral DNA amplification and viral coat protein production during the polydnaviral replication.

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.

A polydnavirus, Cotesia plutella bracovirus (CpBV), encodes host translation inhibitory factors (HTIFs). Two HTIFs have been identified and named as CpBV15α and CpBV15β. HTIFs specifically inhibit host gene expression at post transcriptional level. This suggests that HTIFs can discriminate host mRNAs for their inhibition. To test this hypothesis, we chose a storage protein 1 (SP1) for a target and CpBV15β for non-target genes. Both target and non-target genes were constructed in a cloning vector by cloning their 5'UTR+ORF and in vitro transcribed. The capped partial mRNAs were in vitro translated using rabbit reticulocyte lysate in the presence or absence of HTIFs. Translation of SP1 mRNA was significantly inhibited by the HTIF, but those of CpBV15α and CpBV15β were not. We prepared chimeric mRNAs by exchanging 5'UTR and ORF of both target and non-target genes. Inhibitory activity of HTIFs was dependent on the 5'UTR structure. Target 5'UTR had more complicated secondary structure than non-target 5'UTR in terms of free energy required for unwinding. To identify the target molecule of HTIF to discriminate 5'UTR, an immunoprecipitation using HTIF antiserum was conducted. HTIF was co-precipitated with eIF4A that would be required for unwinding the secondary structure of mRNA. These results suggest that HTIF of CpBV can sequester host eIF4A, any mRNAs possessing complicated secondary structures in their 5'UTRs would be difficult to recruit initiation complex for efficient translation.

Polydnavirus is a mutualistic DNA virus found in some braconid and ichneumonid wasps. Its genome is integrated into host chromosome as a provirus. Its replication occurs at ovarian calyx epithelium during host pupal stage to form episomal viral particles. The viral particles are delivered into hemocoel of the parasitized insect along with eggs during wasp oviposition. Several polydnaviral genomes, which are isolated from the episomal virus particles, have been sequenced and exhibit some gene families with speculative physiological functions. This review presents the viral characteristics in terms of its parasitic physiology. For developing new insect pest control tactics, it also discusses several application strategies exploiting the viral genome to manipulate insect physiology.

내부기생봉의 일종인 프루텔고치별(Cotesia plutellae)이 배추좀나방(Plutellae xylostella)을 대상으로 생물적 방제제로 이용되고 있다. 이 기생봉은 생식기관에 폴리드나바이러스를 공생시키고, 이 바이러스의 존재는 기생봉의 성공적 기생에 필수적이다. 본 연구는 암컷 프루텔고치벌 성장에 따라 플리드나바이러스의 복제시기를 결정하였으며, 또한 교미나 기주 요인에 따른 암컷의 생식 능력을 분석하였다. 기생봉은 발육조건에서 용화 2일째 겹눈과 날개와 같은 성충조직을 발달시키기 시작했으며, 5일째에는 촉각을 포함한 모든 성충조직이 발달되어 우화 직전 단계의 모습을 보였다. 프루텔고치벌 폴리드나바이러스에 대한 다클론성항체에 의한 면역블로팅 분석은 용화 4일째에서 바이러스 복제를 확인할 수 있었다. 바이러스 입자들은 용화 5일째 산란관 내부에서 투과전자현미경으로 관찰되었다. 우화후 난소소관의 길이는 변하지 않았지만, 독샘과 난소받침의 크기는 증가했다. 교미한 암컷은 에서 약 8일관 생존하였으며, 초기 4일동안 집중된( 이상) 산란을 보였다. 미교미 암컷은 교미한 암컷에 비해 낮은 산란을 보였으며, 이 미수정난은 모두 수컷으로 발육하였다. 프루텔고치벌은 배추좀나방과 미국흰불나방(Hyphantria cunea)모두를 기생시킬 수 있었다. 그러나 배추좀나방에서 더 빠르고, 높은 기생율을 보였다.