Biomedical subjects

B A Webb

Publications and source records attributed to B A Webb.

At least 19 recordsLinked to original sources

PubMed2000

A gene encoding a polydnavirus structural polypeptide is not encapsidated.

Polydnaviruses are symbiotic viruses associated with some parasitic Hymenoptera that are vertically transmitted as proviruses within wasp genomes. To study this symbiotic association a gene encoding an abundant Campoletis sonorensis polydnavirus virion protein was characterized. This gene is not encapsidated but resides in the wasp genome where it is expressed only during virus replication. Immunolocalization studies detected the encoded 44-kDa protein only in oviduct tissue with ultrastructural studies detecting epitopes between or on virion envelopes. Expression and localization of the 44-kDa protein are consistent with its being a viral structural protein but localization of the gene only within the wasp genome is atypical, raising the possibility that this protein is adventitiously packaged during virion assembly. To address this possibility, quantitative dot blot and genomic Southern blot hybridizations were performed to determine whether the copy number of the p44 gene increased disproportionately during replication, as would be expected for a gene encoding a virion protein. The copy number of the p44 gene increases in tissues supporting virus replication but is unchanged in other tissues, suggesting that this gene is amplified in replicative cells. The data indicate that genes encoding polydnavirus virion proteins may be distributed between wasp and encapsidated viral genomes.

Amino Acid Sequence↗

PubMed2000

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex.

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.

Actin-Related Protein 2↗

PubMed2000

Parasitism-linked block of host plasma melanization.

When parasitized by the Ichneumonid parasitoid Campoletis sonorensis, larvae of the Noctuid moth, Heliothis virescens, are unable to mount an effective immune response against parasitoid eggs. Defensive melanization of plasma and cellular encapsulation of parasite eggs are dramatically inhibited by infection with the symbiotic immunosuppressive C. sonorensis ichnovirus (CsIV). This study demonstrates that the CsIV-mediated inhibition of melanization is associated with reduction in the enzymatic activity and protein titer of key enzymes in the melanization pathway, phenoloxidase, dopachrome isomerase, and DOPA decarboxylase. Inhibition of the synthesis of key melanization enzymes leads to reductions in the melanization substrates l-dihydroxyphenylalanine, N-acetyldopamine, and N-beta-alanyl dopamine from millimolar to nanomolar levels in parasitized larvae. By contrast, concentration of a precursor catecholamine, dopamine, rises fourfold in these larvae. Thus in CsIV-infected larvae, enzymatic deficiencies in the melanization pathway lead to reduced concentrations of specific enzyme substrates, causing failure of melanization in parasitized insects.

Animals↗

PubMed1999

Cloning and expression of a gene encoding a Campoletis sonorensis polydnavirus structural protein.

Polydnaviruses are the only known group of mutualistic viruses. They are required for successful parasitization in many braconid and ichneumonid parasitoids. The intimacy of this mutualistic association is indicated by the integration and vertical transmission of polydnaviruses in wasp genomes and by their asymptomatic, developmentally regulated replication. The evolution of this mutualism raises several interesting issues that require a better understanding of the viral genome and viral replication. To develop probes for virus replication and morphogenesis, we have begun to characterize several viral structural proteins. A 699 bp cDNA encoding the p12 viral structural protein was cloned and sequenced. The p12 gene localizes to viral segment Y and encodes a predicted protein of 92 amino acids that does not encode a signal peptide and is unrelated to known peptide or nucleic acid sequences. The p12 mRNA is detected at the onset of virus replication. mRNA titers increase with increasing rates of virus replication. Polyclonal antisera raised against histidine-tagged p12 protein expressed in bacteria reacted specifically with the p12 polypeptide in Western blots of CsPDV virions. The p12 polypeptide was not detected in non-replicative wasp or lepidopteran tissues by Western blot analyses but was readily detected in protein extracts of wasp ovaries. The data indicate that the p12 gene is a viral gene encoding a virion protein and provides a specific probe for virus replication that will be useful for studying the evolution of this group of mutualistic viruses.

Amino Acid Sequence↗

PubMed1998

Polydnavirus-mediated inhibition of lysozyme gene expression and the antibacterial response.

Parasitism of lepidopteran host larvae by hymenopteran parasitoids impairs the cellular immune response via expression of polydnavirus genes. Encapsulation of parasitoid eggs is thereby prevented. Parasitized insects are susceptible to opportunistic infections, suggesting that additional components of the immune system are affected. Insects normally respond to infection by inducing the synthesis of an array of antibacterial factors, including cecropins and lysozyme via a NFkappaB/lkappaB-like signal transduction pathway. To characterize the effects of PDVs on the antibacterial immune response, plasma antibacterial activities were assayed in H. virescens larvae infected with the C. sonorensis PDV. Plasma lysozyme activity in Heliothis virescens was reduced in parasitized and PDV-infected larvae after immune challenge. To examine the regulation of lysozyme after CsPDV injection, the Heliothis virescens lysozyme cDNA was cloned. In contrast to plasma lysozyme activity, the 1.1 kb lysozyme mRNA was induced in fat body and haemocytes by known elicitors. The data suggest that CsPDV, like some other viruses, regulates host cell gene expression at the level of translation. We propose that the immunodeficiencies caused by CsPDV injection are caused, in part, by the targeted translational inhibition of specific humoral immune response transcripts.

Amino Acid Sequence↗

PubMed1997

Expression, assembly, and proteolytic processing of Helminthosporium victoriae 190S totivirus capsid protein in insect cells.

The dsRNA genome (5.2 kbp) of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs). The 5' proximal ORF codes for the capsid protein (CP) and the 3' ORF codes for an RNA-dependent RNA polymerase. Although the capsid of Hv190SV is encoded by a single gene, it is composed of two major closely related polypeptides, either p88 and p83 or p88 and p78. Whereas p88 and p83 are phosphoproteins, p78 is nonphosphorylated. Expression of the CP ORF in insect cells generated both p78 and p88 which assembled into virus-like particles. The finding that p78, p83, and p88 share a common N-terminal amino acid sequence is consistent with the determination that N-terminal, but not C-terminal, CP deletions were incompetent for assembly. Evidence was obtained that p78 is derived from p88 via proteolytic cleavage at the C-terminus. Proteolytic processing may play a regulatory role in the virus life cycle since it leads to dephosphorylation of CP and a subsequent decrease in virion transcriptional activity.

Animals↗

PubMed1997

Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae.

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 gene 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.

Animals↗

PubMed1997

Purification and analysis of a polydnavirus gene product expressed using a poly-histidine baculovirus vector.

The VHv1.1 polydnavirus gene has been implicated in suppressing the encapsulation response in parasitized insects [Li and Webb (1994) J. Virol. 68, 7482-7489]. In order to characterize this gene product and to further our analysis of its immunosuppressive function, we expressed the VHv1.1 using a custom-designed C-terminal poly-histidine baculovirus vector which allows for high expression and single-step purification of the protein. The 34 kDa VHv1.1 protein was expressed in baculovirus-infected cell cultures and in H. virescens larvae. Highly enriched preparations of the secreted VHv1.1 protein were obtained after affinity chromatography using a NTA-(Ni2+) resin. Characterization with purified preparations of the VHv1.1 protein established that the protein is N-glycosylated, containing glycogroups which are PNGase F-sensitive but Endo H-resistant. The recombinant VHv1.1 protein bound to hemocytes in vitro and in vivo and was endocytosed in a manner similar to the native protein produced in CsPDV-infected larvae.

Animals↗

PubMed1997

Polydnavirus infection inhibits translation of specific growth-associated host proteins.

The wasp Campoletis sonorensis injects a polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. CsPDV protects the wasp egg and larvae by selectively disabling the host's cellular immune response, and by altering host physiology, growth, and development. Among the changes in host physiology brought about by CsPDV infection is a rapid, and specific decline in the translation of fat body mRNAs encoding selected major plasma proteins. Translational inhibition of the synthesis of all storage protein monomers, p82 (Riboflavin binding hexamer), and p74/p76 (arylphorin), occurs upon infection with CsPDV. Moreover, the prewandering peak of the plasma enzyme juvenile hormone esterase (JHE) was blocked by CsPDV injection. Northern blotting of fat body mRNA demonstrated that transcript levels of storage proteins were not affected by infection. Plasma titers of the iron binding proteins transferrin (p72) and ferritin (p24/26), and of the plasma juvenile hormone binding protein (p25) were not changed by CsPDV infection. That storage protein and JHE synthesis are translationally suppressed, while the synthesis of other plasma proteins continues apace, suggests that CsPDV infection may lead to translational discrimination among available mRNAs in CsPDV infected fat bodies. The effect of this translational discrimination is to shunt host resources away from larval growth and adult development, which presumably makes them available to the developing endoparasitoid.

Animals↗

PubMed1997

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

Promoter activity of the Campoletis sonorensis polydnavirus (CsPDV) WHv1.6 gene was analysed by transient transfection assays in insect cell culture using constructs expressing the CAT gene. Deletions of the WHv1.6 gene promoter were used to define promoter regions important for expression. Progressive deletion of the regions upstream of the TATA box reduced the promoter activity, whereas deletions eliminating the TATA box abolished promoter activity. Cis-activating elements were detected up to 1 kb upstream of the WHv1.6 transcription initiation site (TIS). Promoter elements increasing transcription were detected between -444 and -550 bp and between -831 and -1035 bp relative to the TIS. Analysis of the 3' flanking sequences of the WHv1.6 gene indicated that the polyadenylation signals were the only important elements affecting expression in the constructs. Comparison of promoter regions of four cysteine-rich CsPDV genes revealed homologous sequences that may be important for transcriptional regulation of polydnavirus gene expression in parasitized Heliothis virescens larvae.

Animals↗

PubMed1997

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

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.

Animals↗

PubMed1996

Evaluation of reverse transcriptase polymerase chain reaction for the detection of eastern equine encephalomyelitis virus during vector surveillance.

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was evaluated for the detection of eastern equine encephalomyelitis virus (EEEV). EEEV was detected by amplification of a 416-bp PCR product from within the E2 gene. Internal restriction endonuclease digestion and hybridizations to EEEV RNA demonstrated that the PCR product was amplified from EEEV. PCR amplifications from serial dilutions of an EEEV isolate identified by a neutralization test and titered by an infectious assay in cell culture indicated that this RT-PCR assay detected viral RNA at concentrations below 1 plaque forming unit(PFU) per reaction. The performance of the PCR assay in detection of EEEV was compared with an infectious assay detection procedure (IA/IFA) as part of the New Jersey 1993 vector surveillance program. During 1993, 7,007 field-collected Culiseta melanura (Coquillett) were assayed in 522 pools by both RT-PCR and IA/IFA. EEEV was detected in 95 pools by RT-PCR and 17 pools by IA/IFA; all IA/IFA positive pools were also positive by RT-PCR. During the 1993 field season, RT-PCR consistently detected virus at enzootic foci earlier that IA/IFA and in greater numbers of mosquito pools. The data indicated that viral RNA may be present earlier and in more mosquitoes than indicated by IA/IFA.

Aedes↗

PubMed1996

Isolation and characterization of a member of the cysteine-rich gene family from Campoletis sonorensis polydnavirus.

The endoparasitic wasp Campoletis sonorensis injects a symbiotic polydnavirus into its host Heliothis virescens. Viral gene expression protects the wasp egg and larva from encapsulation by host haemocytes. Three related C. sonorensis polydnavirus (CsPDV) genes, which are expressed in parasitized H. virescens, have been previously isolated and grouped into a cysteine-rich gene family. In this report, a CsPDV gene encoding an abundant 1.4 kb mRNA expressed in parasitized insects was isolated and mapped to viral segment V (15.2 kb) by Southern blotting and PCR. The VHv1.4 cDNA is 1338 bp long and has an ORF that encodes 322 amino acids with two complete and one partial cysteine motifs. Similar to other characterized CsPDV cysteine motifs, the VHv1.4 motifs are also characterized by six cysteines at conserved positions and variable inter-cysteine amino acids. DNA sequence analyses show that the VHv1.4 gene shares regions of significant identity (73-97%) with the VHv1.1 gene, a member of the cysteine-rich gene family. The VHv1.4 and the VHv1.1 proteins are 62% identical overall; at the N termini including the signal peptide and the N-terminal cysteine motif the identity is greater (90%). Unlike other CsPDV cysteine-rich genes, the VHv1.4 cDNA has two conserved domains (77% identical in nucleotides, 55% identical in amino acids) that presumably result from the duplication of a portion of the gene. The VHv1.4 gene has four introns with splicing sites located at positions similar to VHv11.1 introns. Introns 2 and 3, located in the first and second domains respectively, have greater identity (97%) than the flanking exon sequences (77%). We propose, based on the evidence presented in this paper, that the VHv1.4 gene is a new member of the cysteine-rich polydnavirus gene family.

Amino Acid Sequence↗

PubMed1996

Expression of polydnavirus genes under polydnavirus promoter regulation in insect larvae infected with baculovirus recombinants.

We have evaluated the use of baculoviruses to deliver Campoletis sonorensis polydnavirus (CsPDV) genomic DNA into lepidopteran larvae to facilitate the identification of functional CsPDV genes. Genomic fragments consisting of regulatory (promoter) and coding sequences for two CsPDV genes (VHv1.1 and WHv1.6) were used to generate CsPDV-baculovirus recombinants and evaluate the expression of genes under the regulation of the CsPDV promoters. Northern blot and primer extension studies established that CsPDV genes were expressed under the control of their own promoters in these CsPDV-baculovirus recombinants. Transcripts were detected as early as 4 h post-infection indicating that temporal activity of CsPDV promoters was retained. The VHv1.1 gene product as expressed from CsPDV-baculovirus recombinants was identical in size and in functional properties to that produced in CsPDV-infected insects. CsPDV-baculovirus recombinants may be useful for the screening and characterization of polydnavirus genes with functional activities that can only be evaluated in insect larvae.

Animals↗

PubMed1994

Evidence for an early immunosuppressive role for related Campoletis sonorensis venom and ovarian proteins in Heliothis virescens.

Shared epitopes among venom, ovarian, and viral proteins may indicate that related proteins have similar functional roles during parasitization of Heliothis virescens by Campoletis sonorensis. Venom and ovarian proteins are introduced directly into the hemolymph during parasitization where they may target hemocytes or other components of the immune system. Polydnavirus expression has been detected in hemocytes, fat body, and other tissues but has not been detected earlier than 4 h after parasitization. Therefore, effects on hemocytes at times earlier than 4 h may not be caused by polydnavirus proteins synthesized in the parasitized insect. Visualization of hemocyte F-actin with fluorescently labeled phallicidin indicated that a dramatic alteration of plasmatocyte and granulocyte cytoskeletons occurred within 1.5 h after parasitization. The predominant non-viral proteins in the ovary introduced during parasitization were immunologically related to venom and viral envelope proteins. These ovarian proteins persist in the hemolymph. Antisera to the ovarian proteins bound to granulocytes and to plasmatocytes to a lesser degree, suggesting that ovarian proteins may be involved in early suppression of the host's immune response after parasitization.

Animals↗

PubMed1994

Polydnavirus infection inhibits synthesis of an insect plasma protein, arylphorin.

The wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the host's cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).

Animals↗

PubMed1994

Apparent functional role for a cysteine-rich polydnavirus protein in suppression of the insect cellular immune response.

Polydnaviruses suppress the cellular immune response and inhibit growth and development in their lepidopteran host, allowing survival of their endoparasitic hymenopteran host. Characterization of genes disrupting insect physiological systems is a major objective in the study of polydnaviruses. Recently, a cysteine-rich gene family encoding a motif composed of invariable cysteine residues flanking hypervariable intercysteine amino acids was described (S.D. Dib-Hajj, B.A. Webb, and M.D. Summers, Proc. Natl. Acad. Sci. USA 90:3765-3769, 1993). They noted similarities to the positive selection pressure for mutations within the vertebrate major histocompatibility complex (MHC) class II genes and speculated that this class of polydnavirus genes may target and disrupt the insect immune system. To study the functional activity of this family of predicted cysteine-rich proteins, the VHv1.1 gene product was produced from bacterial and baculovirus expression systems. Polyclonal antiserum produced from the bacterial fusion protein reacted with a 30-kDa protein from hemocytes, cell-free plasma, and fat body of parasitized larvae. Immunofluorescence analysis of hemocytes from parasitized insects detected the 30-kDa protein bound to granulocytes and plasmacytes. To assay the functional activity of the 30-kDa VHv1.1 protein, a recombinant baculovirus was constructed allowing in vivo expression of the 30-kDa polydnavirus protein from infected insects. Expression of the VHv1.1 protein from the baculovirus system reduced the encapsulation response to washed wasp eggs relative to controls. The experimental evidence demonstrates that Campoletis sonorensis polydnavirus-infected cells secrete VHv1.1 into the hemolymph, where it binds to hemocytes and is associated with the inhibition of the cellular immune response.

Animals↗

PubMed1993

Structure and evolutionary implications of a "cysteine-rich" Campoletis sonorensis polydnavirus gene family.

For successful parasitization, the female Campoletis sonorensis endoparasitic wasp injects a polydnavirus into its host, Heliothis virescens, during oviposition. Viral gene expression induces immunosuppression and alters development of the host. We report here that three abundantly expressed genes, VHv1.1, WHv1.0, and WHv1.6, describes a polydnavirus "cysteine-rich" gene family which may be important in inducing these host manifestations. These genes have a similar primary gene structure and their proteins contain cysteine motifs characteristic of snail ion-channel ligands, the omega-conotoxins. Like the omega-conotoxins, the intercysteine amino acid residues are hypervariable with only three identical amino acids in all motifs. The conservation of this domain in the three viral genes may reflect an important functional role for these viral proteins in the parasitization of H. virescens. The three genes also contain introns similar in sequence at comparable positions in their 5' untranslated leaders and coding sequences. VHv1.1 contains two cysteine motifs, and each motif is interrupted by an intron at the same position as in the cysteine motifs of WHv1.0 and WHv1.6. Intron 2 sequences of WHv1.0 and WHv1.6 are 92% identical, while the immediately flanking exon sequences encoding the cysteine motifs are only 76% identical. This provides an example of nuclear pre-mRNA introns which are more conserved than flanking exons among members of a gene family.

Amino Acid Sequence↗