An Evidence for Host Translation Inhibitory Factor Encoded in a Polydnavirus, Cotesia glomerata Bracovirus, Genome and Its Expression in Parasitized Cabbage White Butterfly, Pieris rapae

Madanagopal, Nalini,Kim, Yong-Gyun Korean Society of Applied Entomology 2007 Journal of Asia-Pacific Entomology Vol.10 No.4

Polydnavirus is a DNA virus symbiotic to some endoparasitic wasps and plays a critical role in accomplishing successful parasitic life cycle of host wasps. Host translation inhibitory factor (HTIF) has been found in some polydnaviral genomes and performs parasitic functions leading to host immunosuppression and redirecting host nutrient usage to wasp development. The cabbage white butterfly, Pieris rapae, parasitized by a gregarious endoparasitoid, Cotesia glomerata, undergoes several physiological alterations including immune malfunctioning and failure of pupal metamorphosis. C. glomerata possesses its own symbiotic polydnavirus, C. glomerata bracovirus (CgBV). Its genome consisted of at least 12 segments in unequal amounts. Parasitized P. rapae hemolymph contained HTIF-like protein, which was determined through an immunoblotting assay using HTIF antibody of C. plutellae bracovirus (CpBV). RT-PCR using HTIF primers of CpBV produced an HTIF-like gene in P. rapae larvae parasitized by C. glomerata. Also, this HTIF-like gene was encoded in CgBV genome and its partial sequence of CgBV showed highly homology (98.5%) to amino acid sequence of an HTIF of CpBV, called $CpBV15{\alpha}$. These results suggest that a common HTIF-like moiety may be shared among Cotesia-associated bracovirus.

A copy of cystatin from the diamondback moth Plutella xylostella is encoded in the polydnavirus Cotesia plutellae bracovirus

김영태,Rahul Hepat,김용균 한국응용곤충학회 2013 Journal of Asia-Pacific Entomology Vol.16 No.4

Cystatins (CSTs) are reversible and competitive inhibitors of cysteine proteases. Some polydnaviruses encode viral CSTs that have been speculated to play a crucial role in viral pathology. Four CSTs have been reported in the episomal genome of a polydnavirus, Cotesia plutellae (synonymous with C. vestalis) bracovirus (CpBV). These 4 CSTs share high sequence homologies with other bracoviral CSTs. Further sequence analysis showed that 2 of the CpBV-CSTs are identical. The remaining 3 CSTs have been designated CpBV-CST1, CpBV-CST2,and CpBV-CST3. Expression analysis indicated that CpBV-CST2 was not expressed in any stage of Plutella xylostella, either parasitized or non-parasitized by C. plutellae. However, both CpBV-CST1 and CpBV-CST3were expressed in all stages of P. xylostella. Interestingly, these 2 genes were also expressed in non-parasitized P. xylostella in all developmental stages. A CST sequence from the non-parasitized larva was 100% identical with that of CpBV-CST1 for the entire open reading frame (ORF). To understand the role of CpBV-CST1 in viral pathology, the ORF was cloned into a eukaryotic expression vector and transiently expressed in non-parasitized larvae. The in vivo transient expression lasted for at least 4 days. Under this condition,the treated larvae suffered significant suppression in immune responses and in development. These results suggest that CpBV-CSTs play a crucial role in parasitism, altering host immune and developmental processes by interrupting normal interactions between CSTs and cysteine proteases in P. xylostella.

Differential expression profile of genes encoded in a genome segment of Cotesia plutellae bracovirus in a parasitized host, Plutella xylostella

Wael GAD,최재영,제연호,김용균 한국곤충학회 2008 Entomological Research Vol.38 No.1

The Polydnaviruses are an insect DNA virus group. The segmented genome of a polydnavirus is located on the host wasp chromosome as a provirus. After replication, virions are delivered into the parasitized host, where their expression products play significant roles in specific processes of parasitism. However, little is known about how viral gene expression is controlled. In the present study, we tested whether different genes in a genome segment are expressed concomitantly. Cotesia plutellae bracovirus (CpBV) is a polydnavirus mutualistic to an endoparasitoid, Cotesia plutellae (Braconidae: Hymenoptera). Its circular genome segments in viral particles were captured by a transposon containing pUC origin sequence and replicated in Escherichia coli. CpBV-S30 (23.5 kb), one of the large CpBV genome segments, was fully sequenced, and seven open reading frames (ORF) were predicted. We analyzed expression patterns of the seven ORF in the diamondback moth, Plutella xylostella, parasitized by C. plutellae. Transcription analysesindicated that all ORF of CpBV-S30 in the parasitized P. xylostella were expressed from the first day of parasitization and then expression levels decreased over the period of parasitization, except for CpBV-H4, which showed an additional expression peak. In terms of tissue specificity in their expression patterns, all ORF were found to be expressed in the fat body, hemocytes and gut of the parasitized host. Promoter sequence analysis showed that all seven ORF had typical promoter elements, including TATA binding sites, but promoter component numbers and kinds varied. These results suggest that genes in a genome segment of CpBV can differentially express in the parasitized host, presumably by their different promoter components.

A copy of cystatin from the diamondback moth Plutella xylostella is encoded in the polydnavirus Cotesia plutellae bracovirus

Kim, Yeongtae,Hepat, Rahul,Kim, Yonggyun Korean Society of Applied Entomology 2013 Journal of Asia-Pacific Entomology Vol.20 No.1

Cystatins (CSTs) are reversible and competitive inhibitors of cysteine proteases. Some polydnaviruses encode viral CSTs that have been speculated to play a crucial role in viral pathology. Four CSTs have been reported in the episomal genome of a polydnavirus, Cotesia plutellae (synonymous with C. vestalis) bracovirus (CpBV). These 4 CSTs share high sequence homologies with other bracoviral CSTs. Further sequence analysis showed that 2 of the CpBV-CSTs are identical. The remaining 3 CSTs have been designated CpBV-CST1, CpBV-CST2, and CpBV-CST3. Expression analysis indicated that CpBV-CST2 was not expressed in any stage of Plutella xylostella, either parasitized or non-parasitized by C. plutellae. However, both CpBV-CST1 and CpBV-CST3 were expressed in all stages of P. xylostella. Interestingly, these 2 genes were also expressed in non-parasitized P. xylostella in all developmental stages. A CST sequence from the non-parasitized larva was 100% identical with that of CpBV-CST1 for the entire open reading frame (ORF). To understand the role of CpBV-CST1 in viral pathology, the ORF was cloned into a eukaryotic expression vector and transiently expressed in non-parasitized larvae. The in vivo transient expression lasted for at least 4 days. Under this condition, the treated larvae suffered significant suppression in immune responses and in development. These results suggest that CpBV-CSTs play a crucial role in parasitism, altering host immune and developmental processes by interrupting normal interactions between CSTs and cysteine proteases in P. xylostella.

Parasitism by Cotesia glomerata Induces Immunosuppression of Pieris rapae: Effects of Ovarian Protein and Polydnavirus

Madanagopal, Nalini,Kim, Yong-Gyun Korean Society of Applied Entomology 2006 Journal of Asia-Pacific Entomology Vol.9 No.4

A gregarious endoparasitoid wasp, Cotesia glomerata, parasitizes the cabbage butterfly, Pieris rapae. During wandering larval stage for pupal metamorphosis, the parasitoid larvae egress from the parasitized host to form cocoons thus eventually leading to death of the host. This study focused on the effect of C. glomerata parasitization on cellular immune response of P. rapae. For this purpose, an ideal anticoagulant buffer was formulated to procure the hemocytes in native form with morphological, behavioral, and functional characteristics. The hemocytes selectively encapsulated only DEAE beads under in vitro conditions and a quantitative study revealed about 70% of the beads being encapsulated. On the other hand, calyx fluid from C. glomerata injected to P. rapae markedly inhibited the spreading ability of the hemocytes in a dose-dependent manner and also attenuated the in vitro encapsulation response of the hemocytes against the cationic bead. The calyx fluid contained polydnavirus as well as ovarian proteins. The isolated polydnavirus genome consisted of variously sized-segments with their unequal amounts. The P. rapae injected with the calyx fluid expressed several polydnaviral genes within 2 h. These results suggest that the immunosuppression of the parasitized P. rapae may be induced by the polydnaviral gene products as well as ovarian proteins.

Exogenous JH and ecdysteroid applications alter initiation of polydnaviral replication in an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera)

( Bok Ri Park ),( Yong Gyun Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.6

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site (``AGCTTT``) from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication. [BMB reports 2011; 44(6): 393-398]

A SERI technique reveals an immunosuppressive activity of a serine-rich protein encoded in Cotesia plutellae bracovirus

( Karen P. Barandoc ),( Jay Young Park ),( Yong Gyun Kim ) 한국생화학분자생물학회 (구 한국생화학회) 2010 BMB Reports Vol.43 No.4

Polydnavirus genome is segmented and dispersed on host wasp chromosome. After replication, the segments form double-stranded circular DNAs and embedded in viral coat proteins. These viral particles are delivered into a parasitized host along with parasitoid eggs. A serine-rich protein (SRP) is predicted in a polydnavirus, Cotesia plutellae bracovirus (CpBV), genome in its segment no. 33 (CpBV-S33), creating CpBV- SRP1. This study explored its expression and physiological function in the diamondback moth, Plutella xylostella, larvae parasitized by C. plutellae. CpBV-SRP1 encodes 122 amino acids with 26 serines and several predicted phosphorylation sites. It is persistently expressed in all tested tissues of parasitized P. xylostella including hemocyte, fat body, and gut. Its physiological function was analyzed by injecting CpBV-S33 and inducing its expression in nonparasitized P. xylostella by a technique called SERI (segment expression and RNA interference). The expression of CpBV-SRP1 significantly impaired the spreading behavior and total cell count of hemocytes of treated larvae. Subsequent RNA interference of CpBV-SRP1 rescued the immunosuppressive response. This study reports the persistent expression of CpBV-SRP1 in a parasitized host and its parasitic role in suppressing the host immune response by altering hemocyte behavior and survival. [BMB reports 2010; 43(4): 279-283]

Juvenile Hormone-responsive Promoter Element of a Late Expressed Viral gene Encoded in a Polydnavirus, Cotesia plutellae Bracovirus

Rahul P. Hepat,Yonggyun Kim 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.10

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.

Disruption of cell-cell interaction by a polydnavirus gene, CpBV-ELP1

Ahmed Abdel-Fattah,Yonggyun Kim 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05

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.

RNA interference of two ovary transcripts of an endoparasitoid wasp, Cotesia plutellae, suppresses replication of its symbiotic polydnavirus

Bok-ri Park,Yonggyun Kim 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05

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.