Rapid and efficient isolation of highly specific antibodies from an antiserum against a pool of proteins.

Antibodies with desired specificity to proteins of interest provide important and versatile tools for detecting and localizing specific proteins in organisms. With the rapidly increasing number of genes cloned, the demand for antibodies to the gene products is increasing greatly. We developed a procedure to isolate highly specific antibodies to an insect intestinal mucin (IIM) from a polyclonal antiserum, which served as a "library of antibodies," by using an E. coli lysate of the IIM cDNA clone. This procedure allows rapid and efficient isolation of target protein specific antibodies from a polyclonal antiserum made against a pool of antigens.

Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion.

The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9P35AcV5-1 and Sf9P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9P35AcV5-1 and Sf9P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.

Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae).

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.

Molecular structure of the peritrophic membrane (PM): identification of potential PM target sites for insect control.

Peritrophic membranes (PMs) are an invertebrate-unique structure that lines the digestive tract, playing important roles in facilitating food digestion and providing protection to the gut epithelium. The importance of PMs in insects has been recognized ever since its presence was identified 200 years ago. In the last 5 years, significant progress towards understanding the PM molecular structure and the mechanism for PM formation has been made. Recent studies on Type 1 PMs from lepidopteran larvae have suggested a model for the PM molecular structure and formation. The important physiological functions of the PM suggest that PMs can be a significant structural target for insect control and the current understanding of the structure of lepidopteran larval PMs has provided us with potential opportunities for targeting the PM by various mechanisms.

Characterization of a granulovirus from the cassava hornworm (Erinnyis ello: Sphingidae).

A Colombian isolate of Erinnyis ello granulovirus (EeGV) was characterized by electron microscopy, restriction endonuclease digestion, and SDS-PAGE. Electron microscopy showed the occlusion bodies to have a morphology typical of granuloviruses. The restriction patterns of DNA from EeGV and the granuloviruses of Trichoplusia ni (TnGV) and Pieris rapae (PrGV) show little or no similarity, indicating little relatedness among these viruses. EeGV was estimated to possess a relatively small genome of 90.5 +/- 0.5 kbp. SDS-PAGE analysis compared the occulsion body and enveloped nucleocapsid proteins of EeGV and TnGV, and the polypeptide patterns also showed little similarity between these viruses. These analyses, as well as comparison of our results to those reported for other granuloviruses, indicate that EeGV represents a new granulovirus isolate.

Calcofluor disrupts the midgut defense system in insects.

The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection.

Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.

The insect immune protein scolexin is a novel serine proteinase homolog.

Scolexin is a coagulation-provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin-like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism.

Peritrophic membrane structure and formation of larvalTrichoplusia niwith an investigation on the secretion patterns of a PM mucin.

Peritrophic membrane or matrix (PM) secretion and formation patterns were examined in the cabbage looper larvae (Trichoplusia ni[Hubner]) by transmission and scanning electron microscopy (SEM). PM first became visible in the lumen between tips of the microvilli and the stomodeal valves as a single layered fibrous structure that became more compact in appearance in the middle and posterior mesenteron. In the anterior mesenteron, nascent PM was visible within the brush border as a fibrous linear structure that contained both the major PM matrix protein, invertebrate intestinal mucin (IIM) and chitin-containing structures. Even though delamination events were confined to the anterior mesenteron, IIM was secreted by columnar epithelial cells throughout the length of the mesenteron. SEM of the midgut epithelium revealed PM covering individual epithelial cells.

Observations on the presence of the peritrophic membrane in larval Trichoplusia ni and its role in limiting baculovirus infection.

Light microscopical examinations of dissected and stained peritrophic membranes (PMs) were conducted to determine the presence or absence of this protective structure in larvae of Trichoplusia ni, prior to and through ecdysis. Observations of fourth- and fifth-instar larvae of T. ni from two independent rearing colonies showed that PMs were present and lined the midgut prior to, during, and immediately after ecdysis in both instars. Western blot analysis of insect intestinal mucin (IIM), a major protective protein in the T. ni PM, indicated that synthesis of IIM occurred during T. ni embryonic development, or more precisely, that IIM synthesis started approximately 4 h prior to hatching. These results demonstrated that the neonate T. ni midgut is lined with a protective mucinous layer at hatching. A baculovirus enhancin from T. ni granulosis virus (TnGV) enhanced per os viral infections of budded viruses (BVs) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and T. ni single nuclear polyhedrosis virus (TnSNPV) in neonate, fourth-, and fifth-instar larvae of T. ni. These results provided further evidence that the PM may serve as a partial barrier to viruses in the midgut lumen and that enhancins can facilitate the infection process.

Binding and fusion of Autographa californica nucleopolyhedrovirus to cultured insect cells.

Binding of baculoviruses to insect cells and fusion of the virus envelope to cell membranes are early events suggested to be affected by baculovirus enhancins. The binding of Autographa californica nucleopolyhedrovirus (AcMNPV) to the Spodoptera frugiperda cell line Sf21 and the fusion of the virus envelope to cell membranes were characterized. Virus binding assays demonstrated that AcMNPV budded virus (BV) bound to specific binding sites on Sf21 cells with an avidity of 2.3 x 10(10) M(-1). The cells displayed 3.1 x 10(3) specific binding sites per cell in a confluent monolayer. In addition, the effects of pH, buffer composition and cation concentration on the binding were examined. Using a fluorescent probe (R18) and fluorescence microscopy, the fusion of AcMNPV BV envelope to the cell membrane was directly visualized in living cells. It has been reported that Trichoplusia ni nucleopolyhedrovirus enters Sf21 cells by membrane fusion at the cell surface; however, the present studies confirmed the well established concept that adsorptive endocytosis is the major entry pathway for baculovirus BV infection. Membrane fusion kinetics and fluorescence microscopy demonstrated and verified that the envelope-cell membrane fusion was triggered by acidification. The effect of a T. ni granulovirus enhancin on virus binding and membrane fusion was examined, and no increase in activity was observed.

An intestinal mucin is the target substrate for a baculovirus enhancin.

An invertebrate intestinal mucin (IIM) was identified from a lepidopterous insect, Trichoplusia ni. The IIM is a major protein constituent of the peritrophic membrane that facilitates the digestive process, as well as protecting invertebrate digestive tracts from microbial infections. The IIM demonstrated biochemical characteristics similar to vertebrate mucins, but exhibited strong association with the chitin-containing peritrophic membrane matrix. We have demonstrated that a baculovirus enhancin, which is encoded and carried by specific baculoviruses, has mucin-degrading activity both in vitro and in vivo. The in vivo degradation of IIM by enhancin was correlated with the enhancement of baculovirus infections in insects. These findings have shown that viruses have evolved a novel strategy to overcome intestinal mucinous barriers against microorganisms by utilizing a mucin-degrading enzyme.

Molecular cloning and sequencing of a novel invertebrate intestinal mucin cDNA.

The first invertebrate intestinal mucin, termed insect intestinal mucin (IIM), was recently identified from Trichoplusia ni larvae (Wang, P., and Granados, R. R. (1997) Proc. Natl. Acad. Sci. U. S. A. , in press). We report the cDNA cloning and sequencing of IIM, which is only the second completely sequenced intestinal mucin after human intestinal mucin, MUC2. To clone and sequence the cDNA for IIM, a T. ni larval midgut cDNA expression library was constructed and screened with an anti-IIM antiserum. Two full-length cDNA clones for IIM were identified and sequenced. The deduced proteins from the two cDNA clones contained 807 and 788 amino acid residues, respectively. The structural organization of IIM is similar to that of MUC2, containing a 25-amino acid signal leading sequence and two threonine/proline/alanine-rich tandem repeat domains flanked by cysteine-rich sequences. One tandem repeat domain contained two repeating units, TTTQAP and AATTP, and the other contained one repeating unit, TAAP. The cysteine-rich regions showed potential chitin binding features. By immunolocalization in tissue sections, it was determined that IIM is expressed in midgut tissues. The IIM mRNA is abundant in the midgut tissue, and Northern blot analysis indicated that IIM transcripts were not polydispersed as is found in mammalian mucin transcription.

The plasma protein scolexin from Manduca sexta is induced by baculovirus infection and other immune challenges.

Manduca sexta larvae infected per os with a sublethal dose of Erinnyis ello granulosis virus (EeGV) contain an induced plasma protein of ca 33-36 kDa [Finnerty et al. (1994) J. Invertebr. Pathol. 63, 140-144]. This virus-induced protein shares characteristics with scolexin, a bacteria-induced plasma protein from M. sexta, so in this study we compared the two proteins. Two-dimensional gel electrophoresis reveals the induction of polypeptides corresponding to the subunits of scolexin in the plasma from EeGV-infected, as well as bacteria-injected, larvae. Immunoblots of these two-dimensional polyacrylamide gels show that antiserum against the virus-induced protein cross-reacts with the putative scolexin polypeptides induced by either EeGV infection or bacteria injection. Amino acid analysis of the virus-induced protein shows it to be very similar to scolexin, and the N-terminal sequences of the two proteins are nearly identical. From these data, we conclude that the virus-induced protein and scolexin are identical, or at least isoforms of the same protein. Using immunoblot, we also demonstrate that scolexin is induced in M. sexta plasma by injection with yeast or lipopolysaccharide from Serratia marcescens.

Enhancin, the granulosis virus protein that facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease.

Enhancin is a Trichoplusia ni granulovirus protein that facilitates nucleopolyhedrovirus (NPV) infections in lepidopterans. Gel filtration and ion exchange chromatography and immobilized alpha-macroglobulin were used to purify this protein and the removal of the contaminating proteases did not diminish the in vivo or in vitro activity of enhancin. Metal chelators were the only protease inhibitors capable of preventing digestion of the peritrophic membrane (PM) proteins by enhancin, indicating that enhancin is a metalloprotease. In addition, the canonical zinc binding site, HEXXH, found in most metalloproteases, was identified in the sequences of enhancins from three different granuloviruses. The identity of enhancin as a metalloprotease that facilitates NPV infections in lepidopterous larvae was confirmed by the expression of enhancin in a recombinant Autographa californica MNPV-baculovirus system and the purification of a recombinant enhancin that was active in neonate bioassays and that digested specific PM proteins. The recombinant enhancin was also inhibited by metal chelators and both the native and recombinant enhancin could be reactivated by divalent ions, further confirming that enhancin is a metalloprotease.

Physical mapping and identification of interspersed homologous sequences in the Trichoplusia ni granulosis virus genome.

A restriction fragment library representing 89.3% of the genome of Trichoplusia ni granulosis virus (TnGV) was constructed. The library consisted of 13 of the 16 BamHI fragments, 18 of the 22 EcoRI fragments, and 6 of the 27 PstI fragments. By restriction endonuclease and Southern blot analysis of cloned or genomic viral DNA fragments, a complete physical map of TnGV was constructed for BamHI, EcoRI, PstI and XhoI. Three interspersed homologous regions (ihs1-ihs3) were identified from hybridization experiments and sequenced. Each TnGV ihs has an approximate size of 400 bp and shows homology to the other two. The orientation of ihs2 is inverted relative to ihs1 and ihs3. TnGV ihs regions do not have repetitive motifs or palindromic sequences, in contrast to homologous regions (hrs) of nuclear polyhedrosis viruses (NPVs). The genomic locations of TnGV ihs1-ihs3, represented in percentage map units, were very similar to those of ihs sequences previously reported in Bombyx mori NPV, suggesting that the ihs may be a novel type of cis-acting element common among baculoviruses. Additionally, an inverted repeat sequence, having overlapping, multiple inverted repeats of 400 bp, was identified to the left of ihs3 on the linearized genome map of TnGV.

Characterization of the Helicoverpa armigera and Pseudaletia unipuncta granulovirus enhancin genes.

Enhancins are baculovirus proteins capable of enhancing infections in insect larvae by other baculoviruses. We have identified the enhancin proteins in four species of granulovirus (GV). In this paper we describe the cloning and sequencing of the enhancin genes of the Pseudaletia unipuncta granulovirus-Hawaiian strain (PsunGV-H) and the Helicoverpa (Heliothis) armigera granulovirus (HearGV). The PsunGV-H enhancin gene is virtually identical to the previously characterized Trichoplusia ni GV (TnGV) enhancin gene. In contrast, a comparison of the predicted amino acid sequences of TnGV enhancin (901 amino acids) and HearGV enhancin (902 amino acids) revealed an overall identity of only 80%, with greater conservation (88%) from amino acids 1-550. Primer extension analysis of enhancin RNAs identified the baculovirus late promoter motif that serves as the transcriptional start site in the HearGV enhancin gene. It is located three nucleotides from the putative enhancin translational initiator codon. RNase protection analysis demonstrated that both read-through and termination occur at the 3' end of the gene. Since a partial open reading frame (ORF) was identified immediately downstream of the 3' end of the enhancin ORF, these data suggested that a sizeable fraction of the enhancin mRNAs may be bi-cistronic and share a common 3' end with a downstream transcription unit.

Interaction of Trichoplusia ni granulosis virus-encoded enhancin with the midgut epithelium and peritrophic membrane of four lepidopteran insects.

Enhancin, an infectivity-enhancing protein from Trichoplusia ni granulosis virus (TnGV) was tested for its ability to increase Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) infection in the larvae of four lepidopteran insects. Enhancin increased the mortality of AcMNPV infection in all the four insect species tested. Peritrophic membrane (PM) assays showed altered protein profiles in PMs treated with enhancin in all the four species. This supports the hypothesis that enhancin affects virus infection by altering the structural integrity of the PMs. The binding of enhancin to the midgut brush border membranes (BBMs) was determined and specific binding sites were found on the BBM of Pseudaletia unipuncta. No specific binding sites were found on the BBMs of T. ni, Helicoverpa zea or Spodoptera exigua. Therefore, specific binding of enhancin to the midgut cell membrane may not be necessary for the enhancement of baculovirus infection in insects.

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein.

The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinant Autographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established from Spodoptera frugiperda, Trichoplusia ni, and Mamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in the M. brassicae-derived MB0503 cell line was Endo H resistant. The commonly used S. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.