Promoter analysis of a cysteine-rich Campoletis sonorensis polydnavirus gene.

Cuit, L; Webb, Bruce A.

doi:10.1099/0022-1317-78-7-1807

Cited by 12 publications

(6 citation statements)

References 40 publications

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“…Analyses of the VHv1.4 and VHv1.1 mRNAs revealed their similarity in tissue-specific and temporal expression patterns, implying that they might be coordinately regulated (Cui and Webb, 1996;Cui and Webb, 1997). Both genes are expressed early and at high levels throughout the entire course of parasitization.…”

Section: Discussionmentioning

confidence: 99%

Expression and hemocyte-targeting of aCampoletis sonorensis polydnavirus cysteine-rich gene inHeliothis virescens larvae

Cui

Soldevila

Webb

1997

Arch. Insect Biochem. Physiol.

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The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine‐rich polydnavirus genes have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine‐rich Vhv1.4 protein. Full‐ length and truncated Vhv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the Vhv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The Vhv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The Vhv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the Vhv1.4 protein is glycosylated at multiple N‐glycosylation sites. Immunofluorescence assays showed that the Vhv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the Vhv1.4 protein was internalized, probably by endocytosis. Specific binding of the Vhv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response. Arch. Insect Biochem. Physiol. 36:251–271, 1997. © 1997 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

Expression and hemocyte-targeting of aCampoletis sonorensis polydnavirus cysteine-rich gene inHeliothis virescens larvae

Cui

Soldevila

Webb

1997

Arch. Insect Biochem. Physiol.

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“…Segment nesting differentially increases the copy number VHv1.4 and WHv1.6 genes (located on many nested segments) relative to their homologs found only on the parental segments (VHv1.1 and WHv1.0 on V and W, respectively). Since CsPDV does not appear to replicate in parasitized insects (42) and CsPDV promoters are of the early or constitutive type (9), the level of gene expression may depend largely on gene copy number. Therefore, segment nesting may be important for the generation of high copy number of functional genes in parasitized insects (14).…”

Section: Discussionmentioning

confidence: 99%

Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family

Cui

Webb

1997

J Virol

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Polydnaviruses (PDVs) are double-stranded DNA viruses with segmented genomes that replicate only in the oviducts of some species of parasitic wasps and are required for the successful parasitization of lepidopteran insects. PDV DNA segments are integrated in the genomes of their associated wasp hosts, and some are nested; i.e., smaller segments are produced from and largely colinear with larger segments. To determine the internal structure of nested viral segments, the first complete nucleotide sequence of a PDV genome segment and its integration locus was determined. By restriction mapping, Southern blot, and sequence analyses, we demonstrated that the Campoletis sonorensis PDV segment W is integrated into wasp genomic DNA. DNA sequence analysis revealed that proviral segment W terminates in two 1,185-bp direct long terminal repeats (LTRs) in the wasp chromosome, while only one LTR copy is present in the extrachromosomal (viral) W. The results suggest that terminal direct repeats are a general feature of PDV DNA segment integration but that the homology and size of the repeats can vary extensively. Segment W contains 12 imperfect direct repeats of six different types between 89 bp and 1.9 kbp with 65 to 90% homology. The orientation and structure of the repeats suggest that W itself may have arisen through sequence duplication and subsequent divergence. Mapping, hybridization, and sequence analyses of cloned R and M demonstrated that these segments are nested within segment W and that internal imperfect direct repeats of one type are implicated in the homologous intramolecular recombination events that generate segments R and M. Interestingly, segment nesting differentially increases the copy number of genes encoded by segment W, suggesting that the unusual genomic organization of PDVs may be directly linked to the unique functions of this virus in its obligate mutualistic association with parasitic wasps.Insects and viruses. The wasp C. sonorensis was maintained on its host Heliothis virescens larvae, and virus and viral DNA were isolated from the female wasp ovaries as described previously (30).Cloning and mapping of viral segments. Viral DNA was separated on 0.7% agarose gel in TAE buffer, and bands of superhelical viral DNA segments R and M were isolated (2, 5). DNA segments were purified by using Geneclean (Bio 101), digested with a restriction enzyme (BamHI, PstI, or XbaI), and cloned into compatibly digested plasmid pZero-1 (Invitrogen). Since some DNA bands contain comigrating but different viral DNA segments, the resulting clones were screened for hybridization to labeled W. Hybridizing clones were mapped with restriction enzymes (27), and subclones were generated in pZero-1 for detailed mapping and sequencing.Wasp genomic DNA isolation and genomic library. Wasp genomic DNA was extracted from pooled male wasps by using a modified procedure (46). A C. sonorensis genomic library was constructed by ligating BamHI-digested GEM-11 (Promega) arms to size-selected, Sau3AI-digested wasp genomic DNA (27). Genomi...

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“…A 250-bp AsNOS fragment was amplified by 35 cycles of PCR (94°C for 40 sec, 53°C for 40 sec, and 72°C for 40 sec) with the primers 5Ј-ACATCAAGACGGAAATGGTTG 3Ј and 5Ј-ACAGACGTAGATGTGGGCCTT 3Ј; for replicates of tissue-specific assays, a 180-bp AsNOS fragment was amplified using identical conditions and the nested primer pair 5Ј-ACTGCAAGATTCCCAAGGTAT 3Ј and 5Ј-ATTCCGC-CTCTTTGAGGGCAA 3Ј. Transcript abundance was normalized against a 300-bp ␤-actin fragment amplified by using the described PCR conditions for 15 cycles with primers designed against A. gambiae ␤-actin (12). For tissue-specific assays, AsNOS transcript abundance was normalized against a 460-bp ribosomal S7 protein gene fragment amplified using 25 cycles of PCR (94°C for 30 sec, 55°C for 30 sec, and 72°C for 40 sec) and published primers (13,14).…”

Section: Rna Isolation and Analysis By Reverse Transcriptase-pcr (Rt-mentioning

confidence: 99%

The mosquitoAnopheles stephensilimits malaria parasite development with inducible synthesis of nitric oxide

Luckhart

Vodovotz

Rosenberg

1998

Proc. Natl. Acad. Sci. U.S.A.

379

386

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We have discovered that the mosquito Anopheles stephensi, a natural vector of human malaria, limits parasite development with inducible synthesis of nitric oxide (NO). Elevated expression of A. stephensi NO synthase (NOS), which is highly homologous to characterized NOS genes, was detected in the midgut and carcass soon after invasion of the midgut by Plasmodium. Early induction is likely primed by bacterial growth in the blood meal. Later increases in A. stephensi NOS expression and enzyme activity occurred at the beginning of sporozoite release. Circulating levels of nitrite͞ nitrate, end-products of NO synthesis, were significantly higher in Plasmodium-infected mosquitoes. Dietary provision of the NOS substrate L-arginine reduced Plasmodium infections in A. stephensi. In contrast, dietary provision of a NOS inhibitor significantly increased parasite numbers in infected mosquitoes, confirming that A. stephensi limits Plasmodium development with NO.

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Promoter analysis of a cysteine-rich Campoletis sonorensis polydnavirus gene.

Cited by 12 publications

References 40 publications

Expression and hemocyte-targeting of aCampoletis sonorensis polydnavirus cysteine-rich gene inHeliothis virescens larvae

Expression and hemocyte-targeting of aCampoletis sonorensis polydnavirus cysteine-rich gene inHeliothis virescens larvae

Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family

The mosquitoAnopheles stephensilimits malaria parasite development with inducible synthesis of nitric oxide

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