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M D Summers

Publications and source records attributed to M D Summers.

At least 19 recordsLinked to original sources
PubMed1992

Stimulation of polydnavirus replication by 20-hydroxyecdysone.

During oviposition the endoparasitic wasp Campoletis sonorensis, introduces a polydnavirus into parasitized insects where viral gene expression is required for endoparasite survival. The polydnavirus is integrated into wasp chromosomal DNA and replicates only in the ovary. Ecdysteroids regulate the developmental expression of many insect genes and may regulate polydnavirus replication. Direct verification of viral replication was performed by dot blot hybridization and by amplifying DNA sequences containing the viral integration site; this 'junction' fragment cannot be amplified from integrated virus. Thoracic ligations and in vitro ecdysteroid treatments of wasp ovaries support the hypothesis that polydnavirus DNA replication is regulated by ecdysteroid during parasite development.

Animals↗
PubMed1992

Sequence, genomic organization of the EcoRI-A fragment of Autographa californica nuclear polyhedrosis virus, and identification of a viral-encoded protein resembling the outer capsid protein VP8 of rotavirus.

We present the sequence and genomic organization of the EcoRI-A fragment of the Autographa californica multicapsid nuclear polyhedrosis virus, which represents 11% of the AcMNPV genome. Fifteen putative open reading frames and their respective amino acid sequences are described. One open reading frame is similar to the VP8 protein of rotavirus.

Amino Acid Sequence↗
PubMed1992

Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate-early 1 transcriptional regulatory protein.

Autographa californica multicapsid nuclear polyhedrosis virus-infected insect cells express a viral immediate-early transcriptional regulatory protein, IE1, that has been shown by transient-expression assays to stimulate the expression of certain baculovirus delayed-early (DE) promoters and to inhibit the expression of other immediate-early (IE) genes. It is believed that certain DE promoters are activated, in part, by direct interactions between IE1 and enhancer elements located in regions adjacent to these genes. We have used transient cotransfection and DNA-binding assays to examine the function of mutant forms of IE1. Our results indirectly show that IE1 has at least two separable domains that are essential for its role in the modulation of baculovirus gene expression. A domain rich in acidic residues and essential for transactivation is located within the N-terminal 145 amino acids of the polypeptide. A second domain, located in the C-terminal 437 amino acids of IE1, is required for inhibitory and DNA-binding activities. Several nontransactivating IE1 mutants trans-dominantly interfered with wild-type IE1 transactivation of enhancer-linked DE genes. trans-dominant interference was expressed only by IE1 mutants that retained the N-terminal putative acidic activation domain, suggesting that this region may be involved in associations with a factor(s) essential for activation of enhancer-linked genes.

Amino Acid Sequence↗
PubMed1991

Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host.

The polydnavirus Campoletis sonorensis virus (CsV) is present in the oviducts of all adult C. sonorensis female wasps and appears to be required for these wasps to parasitize hosts successfully. Physical mapping, Southern blot analysis, and nucleotide sequence analysis demonstrate that the viral DNA B-specific sequences in cloned wasp DNA are colinear with viral genomic segment DNA B from nucleocapsids and are covalently linked to nonviral wasp sequences. Integrated DNA B terminates in 59-nucleotide imperfect direct repeats, but a single repeat exists in the extrachromosomal superhelical viral DNA B. Sequences near each junction form imperfect inverted repeats with sequences near the ends of an internal viral 540-base-pair repeat element gene. CsV appears to be the first documented integrated, nonretroviral DNA virus of insects and probably is vertically transmitted as a provirus.

Animals↗
PubMed1991

Identification of spliced baculovirus RNAs expressed late in infection.

Previous to this study, the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was known to express only one spliced RNA (spliced IE1 or IE0). We have conducted an analysis of RNA expressed during infection of Spodoptera frugiperda cells with AcMNPV and have identified a set of five additional spliced RNAs expressed late in infection. A reverse transcription-polymerase chain reaction analysis was used to confirm the identification of the LS (late, spliced) RNAs. S1 nuclease and primer extension analyses were used to map the transcription initiation sites of LS RNAs. LS1 and LS2 initiated at positions -138 and -117, respectively (relative to the IE0 +1 transcription start site). Both LS1 and LS2 contain an additional cistron potentially encoding a small, highly basic polypeptide. LS3 (-79), LS4 (-22), and LS5 (+51/52) RNAs encode only the predicted downstream IE0 ORF. Although several baculovirus late gene consensus transcription initiation sites (ATAAG) were identified within this region, only LS5 initiated at one of these conserved motifs. An S1 nuclease analysis was done to determine whether unspliced precursors of LS RNAs could be detected. Early in infection, a greater proportion of IE0 RNA was detected in the spliced form; however, during the late phase of infection a significantly greater amount of unspliced precursor forms of LS RNAs was observed.

Amino Acid Sequence↗
PubMed1991

Molecular analysis of a baculovirus regulatory gene.

To better understand the structure and function of a baculovirus regulatory gene, the nucleotide sequence of IE-N expressed by Autographa californica nuclear polyhedrosis virus was determined. The 2.0-kb PstI-EcoRV restriction fragment (97.5 to 98.9 mu) encodes the upstream regulatory sequences, open reading frame, and downstream sequences of the immediate early IE-N gene. Using a convenient restriction site, the 285-bp promoter of IE-N was divided into two functional regions as defined by transient expression assays of mutant sequences. The sequences of IE-N from -1 to -45 nt encoded a minimal promoter capable of directing low levels of transcription. The minimal promoter was fully responsive to positive regulation by IE-N. The upstream region from -46 to -285 nt contains two direct repeats which increased levels of IE-N gene expression. Computer-assisted translation of the IE-N sequence indicates that this fragment of DNA encodes a single long open reading frame with a predicted molecular weight of 47,000. The amino acid sequence of the predicted protein exhibits three motifs common to transcriptional regulators: a serine-threonine rich region, a proline-rich region, and a polyglutamine tract. IE-N autoregulates its own expression and stimulates both IE-1 and IE-0 in transient assays. The stimulation of IE-1 may account for the augmenting activity of IE-N in the IE-1-mediated trans-activation of the 39K promoter.

Amino Acid Sequence↗
PubMed1991

Diagnostic potential of baculovirus-expressed rubella virus envelope proteins.

The envelope glycoproteins E1 and E2 of rubella virus were abundantly expressed in Spodoptera frugiperda Sf9 insect cells by using a baculovirus expression vector. The recombinant protein products were purified by immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The purified recombinant antigen consisted of the envelope polypeptides, corresponding to the viral E1 and E2 proteins, and a polyprotein precursor (molecular mass, 90 to 95 kDa). The antigen was reactive with human convalescent-phase sera in immunoblot analysis, and the reactivity correlated well (r = 0.861) with that of a whole-virus antigen when tested by EIA by using a total of 106 rubella virus immunoglobulin G-positive and -negative serum specimens. When the sera from patients with recent rubella virus infection were tested with the recombinant glycoproteins by EIA, the correlation was not as close (r = 0.690). However, all of the 26 serum specimens were reactive with the recombinant antigen. The results demonstrate that these bioengineered antigens have a potential for use in routine diagnostic assays of rubella virus immunity and recent infection.

Animals↗
PubMed1991

Novel regulatory properties of the IE1 and IE0 transactivators encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus.

The baculovirus Autographa californica multicapsid nuclear polyhedrosis virus expresses two immediate-early genes from the HindIII-G region (map units 90.4 to 96.8) of the genome. During the early phase of infection, nonspliced 1.9-kb and spliced 2.1-kb transcripts are expressed which encode the IE1 and IE0 (spliced IE1) gene products, respectively. These two gene products differ only in that IE0 contains an additional 54 amino acids at the amino terminus. RNA analysis of these two genes during infection revealed that they were differentially expressed. IE1 was expressed early and late, whereas IE0 was expressed only early in infection. The regulation of these two immediate-early genes was analyzed by transient expression assays. The IE1 gene product stimulated expression of IE1 promoter-directed expression but down-regulated expression from the IE0 promoter. The IE0 gene product also transactivated the IE1 promoter but did not affect expression from its own promoter. Unlike IE1, which transactivates the delayed early 39K gene in the presence and absence of the homologous region (hr) enhancers, IE0 transactivated the 39K promoter only in the presence of cis-linked hr5 enhancer. The results of this study in conjunction with previous results suggest that the IE1 gene encodes a multifunctional gene product that may be involved in (i) repression of immediate-early gene expression, (ii) continued expression of its own gene product during infection, and (iii) transactivation of the delayed early and late classes of genes.

Animals↗
PubMed1991

Transient expression of the Autographa californica nuclear polyhedrosis virus immediate-early gene, IE-N, is regulated by three viral elements.

Autographa californica nuclear polyhedrosis virus (AcMNPV) is a double-stranded DNA virus that expresses several immediate-early genes under the control of different promoters. The expression of one of these transcription units, IE-N, is shown here, by a transient expression assay, to be regulated by both cis- and trans-acting viral elements. The steady-state levels of IE-N mRNA were very abundant soon after infection but were nearly undetectable during the late phase of the viral life cycle. Analysis of the transient expression of a reporter construct driven by the IE-N promoter (IE-NCAT) was conducted to define viral elements which regulate IE-N gene expression. Viral enhancer hr1 and two immediate-early genes, IE-1 and IE-N, were shown to affect relative levels of reporter enzyme activity produced by IE-NCAT. The hr1 enhancer stimulated the expression of IE-NCAT, independent of orientation and position relative to the promoter and in the absence of any trans-acting viral factors. Regulation of IE-NCAT expression by the IE-1 and IE-N genes required less than 290 bp of promoter sequences upstream of the site of transcription initiation and was not dependent upon the hr1 enhancer. Coexpression of the IE-N gene had an autostimulatory effect upon IE-NCAT activity, whereas coexpression of the IE-1 gene reduced levels of reporter activity. The levels of reporter activity measured upon coexpression of either immediate-early gene with IE-NCAT linked to the hr1 enhancer appear to be the combined result of both cis- and trans-regulatory elements influencing expression from IE-NCAT. These results suggest that IE-N gene expression in baculovirus infection may be influenced by the concerted activity of three AcMNPV regulatory elements.

Animals↗
PubMed1991

Polyhedrin initiator codon altered to AUU yields unexpected fusion protein from a baculovirus vector.

A recombinant baculovirus expression vector was constructed to express the core (capsid) protein of the hepatitis B virus. Along with the expected 21-kDa polypeptide, a second 24-kDa protein was observed. Immunoprecipitation and immunoblotting using a rabbit polyclonal anticore antiserum demonstrated that the two proteins were related. The core gene originally was cloned in-frame with the polyhedrin initiator codon that had been altered to AUU as a means of preventing fusion protein formation. A transient expression assay revealed expression of the 24-kDa protein was prevented if a frame-shift mutation was created upstream of the HBV core translation start site. These results suggest that the 24-kDa protein was the result of an unexpectedly high level of translation initiation at the AUU codon that gave rise to a polyhedrin-HBV core fusion protein. The 24-kDa core protein was shown to be a polyhedrin fusion protein by immunoblotting with an antipolyhedrin antiserum, and initiation at the AUU was demonstrated by amino terminal protein sequencing. Methods to prevent undesired fusion protein expression using this or similar vectors are discussed.

Amino Acid Sequence↗
PubMed1990

Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells.

Cell lines established from the Lepidopteran insect Spodoptera frugiperda (e.g., Sf9) are used routinely as hosts for the expression of foreign proteins by baculovirus vectors. Previously, we showed that human tissue plasminogen activator (t-PA) was expressed, N-glycosylated, and secreted by Sf9 cells infected with a recombinant baculovirus (Jarvis DL, Summers MD: Mol Cell Biol 9:214-223, 1989). We also showed that t-PA secretion was blocked by tunicamycin (TM), an inhibitor of N-glycosylation, but not by castanospermine (CS) or N-methyldeoxynojirimycin, inhibitors of the initial steps in N-linked oligosaccharide processing. This suggested that the addition, but not the processing, of N-linked oligosaccharides is required for the secretion of recombinant t-PA from baculovirus-infected Sf9 cells. In this study, we present a more generalized evaluation of the role of N-glycosylation in the transport of recombinant glycoproteins through the Sf9 cell secretory pathway. Several different secretory or membrane-bound glycoproteins were expressed in control, TM-treated, or CS-treated Sf9 cells, and their appearance in the medium or on the cell surface was measured. The results showed that TM blocked the transport of some, but not all, of these proteins, whereas CS did not block the transport of any. This suggests that N-glycosylation is sometimes required for the transport of recombinant glycoproteins through the Sf9 secretory pathway, while processing of the oligosaccharides is not. At least two other proteins, p80 and p31, consistently coimmunoprecipitated with the nonglycosylated precursors of recombinant glycoproteins expressed in TM-treated Sf9 cells. Neither was antigenically related to any of the recombinant proteins. Relatively larger amounts of p80 and p31 were coprecipitated when transport was completely blocked by TM compared to when transport was only reduced or was unaffected. These results suggest that p80 and p31 block the transport of some nonglycosylated glycoprotein precursors in TM-treated Sf9 cells by binding to them and producing transport-incompetent heterooligomeric complexes. If this speculation is correct, then p80 and p31 are functionally analogous to the mammalian immunoglobulin heavy chain binding/glucose-regulated 78 kilodalton protein (BiP/GRP78).

Alkaloids↗
PubMed1990

Sequence comparison of cellular and viral copies of host cell DNA insertions found in Autographa californica nuclear polyhedrosis virus.

The nucleotide sequence and structural characteristics of two nonhomologous host DNA insertions of Spodoptera frugiperda (fall armyworm) origin isolated from few polyhedra mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) have been determined. Neither of the host insertions contain long open reading frames suggesting that cellular genes were not introduced into the viral genome. One of the host DNA insertions, IFP1.6, terminated in short imperfect inverted repeats flanked by a duplication of the target sequence, TTAA. Analysis of three cellular copies of this host insertion isolated from a lambda genomic library of S. frugiperda DNA revealed the termini to be highly conserved and also flanked by the same TTAA sequence. IFP1.6 was shown to share homology with a putative host insertion described in the genome of a baculovirus exhibiting wild-type plaque morphology. The second of the host insertions, IFP2.2, had a structure unique among host insertions described in baculoviruses. It lacks terminal repeats but is flanked by duplications of the 8-bp target site sequence. The cellular copy of this insertion was conserved in comparison to the viral copy and was also flanked by a direct 8-bp repeat. This is the first report of the analysis of cellular copies of host DNA insertions frequently associated with baculovirus FP mutants.

Base Sequence↗
PubMed1990

Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells.

Baculoviruses are currently used as vectors for the transient high-level expression of foreign gene products in insect cells. In this study, we demonstrate that baculoviruses can also be made to continuously express a foreign gene product by using the promoter from IE1, an immediate early viral gene, to produce stably-transformed insect cells. This approach gave levels of foreign gene expression lower than those usually obtained with the lytic baculovirus expression vector system. Expression, however, was continuous and stable, and a complex human glycoprotein (tissue plasminogen activator) was processed more efficiently. We conclude that stable transformation is a feasible approach for baculovirus-mediated foreign gene expression in lepidopteran cells, particularly for products that are relatively poorly-expressed and/or processed in lytically infected cells.

Animals↗
PubMed1990

Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences.

Endoparasitic wasps of lepidopteran insects must induce changes in host immunity and development to survive. Depending on the species, this may require wasp venom proteins and/or a polydnavirus. We describe an immunological and genetic relationship between the Campoletis sonorensis polydnavirus and the wasp's venom gland. Monoclonal antibodies raised against venom glands recognized epitopes conserved on several polydnavirus proteins and on multiple wasp oviduct and venom proteins. The viral envelope proteins had molecular masses of 16, 20, 45, and 50 kDa, while a complex of at least five immunoreactive venom-gland and soluble oviduct proteins ranged in size from 24 to 36 kDa. Since the conserved epitopes were present on the viral envelope, neutralization assays were performed. Monoclonal antibodies added to purified virus blocked the normal viral inhibition of host growth and development. To determine whether venom mRNA and viral genes were also related, venom-related cDNA clones were isolated from the wasp oviduct with a venom-gland cDNA probe. Venom-related viral clones were then identified and selected from a viral genomic library and from a parasitized Heliothis virescens cDNA library. Venom-related mRNAs were expressed in the venom gland, the oviduct, and the parasitized host. We propose that the immunological relationship between venom and viral proteins, and the hybridization of venom and viral genes, may reflect an evolutionary relationship in which venom gene homologs were incorporated into the viral genome, thereby allowing viral expression of venom-related genes and enhancing parasite survival.

Animals↗
PubMed1990

Translocation and cleavage of rubella virus envelope glycoproteins: identification and role of the E2 signal sequence.

The structural proteins of rubella virus (RV) are translated as a large polyprotein precursor, p110, which is processed to produce the mature virion components, the 33K capsid protein (C) and the two envelope glycoproteins, E1 (58K) and E2 (42K to 47K). The precise processing mechanism has not been elucidated; however it must include at least two proteolytic cleavages to release the individual virion components from the polyprotein, and it must provide for their dichotomous intracellular distribution. The C protein remains in the cytoplasm where it participates in the formation of nucleocapsids, while the envelope glycoproteins enter the cellular secretory pathway and are N-glycosylated and cleaved. Sequence analysis of the 24S mRNA encoding the polyprotein precursor suggests that both E1 and E2 are preceded by signal peptides for translocation across the membrane of the rough endoplasmic reticulum. A recent study has provided direct evidence that the putative signal peptide preceding E1 can in fact mediate translocation of E1. In this study, we have used in vitro translation-translocation assays to examine further the processing of RV glycoproteins. We have shown that the putative signal sequence preceding E2 can mediate translocation of the E2 protein in the absence of an intact E1 signal peptide. The experiments also revealed that cleavage of the E2-E1 polyprotein requires (i) the E2 signal peptide, (ii) microsomal membranes and (iii) sequences beyond the proximal half of the E1 signal peptide. Together these results suggest that separation of the E2 signal sequence as well as the proteolytic cleavage of E1 from E2 is performed by the cellular enzyme, signal peptidase.

Amino Acid Sequence↗
PubMed1989

Baculovirus polyhedrin promoter-directed expression of rubella virus envelope glycoproteins, E1 and E2, in Spodoptera frugiperda cells.

To study the capability of Spodoptera frugiperda (fall armyworm; Sf9) cells to synthesize and process mature rubella virus (RV) proteins, a cDNA encoding the structural envelope glycoproteins, E1 (58 kDa) and E2 (42-47 kDa) were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during infection under the transcriptional regulation of the polyhedrin gene promoter. By immunoblot analysis with antibodies directed against purified RV, the individual proteins E1 and E2, and human convalescent serum, a polyprotein precursor migrating with an apparent molecular weight of 90-95 kDa was identified in Sf9 cells infected with the recombinant baculovirus, Ac701-RVE. In addition, two proteins migrating somewhat faster than authentic viral E1 and E2 were resolved. Pulse-chase labeling experiments in the absence and presence of tunicamycin, as well as treatment of the recombinant proteins with endo-beta-N-acetyl-D-glucosaminidase H indicated that the recombinant proteins are glycosylated and that the E1 and E2 apoproteins, respectively, were similar in size as compared to their in vitro synthesized counterparts. The recombinant protein products were further detected by some monoclonal antibodies directed against RV. The results presented here indicate that a polyprotein containing the envelope glycoproteins of RV is expressed and proteolytically cleaved in lepidopteran insect cells to form two proteins which resemble authentic E1 and E2. The baculovirus may therefore be suitable for the abundant expression of RV antigen.

Animals↗
PubMed1989

Location and nucleotide sequence of the 25K protein missing from baculovirus few polyhedra (FP) mutants.

Wild-type and few polyhedra (FP) mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were studied to identify and sequence the gene encoding the 25-kDa (25K) protein normally present in AcMNPV-infected Spodoptera frugiperda cells but which is often missing from FP mutant-infected cells. Our previous study had mapped two overlapping late transcripts to the insertion site of host cell DNA within the HindIII-I fragment (33.8 to 37.7 map units) of wild-type AcMNPV. An FP mutant, AcFP875-2, had a 1.6-kbp insertion of S. frugiperda DNA near the 5' end of these transcripts which by S1 analysis were shown to initiate within the host cell sequence. Primer extension analysis revealed that the transcription start for this gene in wild-type virus occurred within a conserved 12-base sequence found near the transcription start sites of several baculovirus late and hyper-expressed genes. A similar 12-base sequence was found at the transcription start site within this 1.6-kbp pair host cell DNA sequence in AcFP875-2. mRNAs from wild-type virus-infected cells were hybridization-selected using a 542-bp SalI subfragment of the 3.2-kbp EcoRI-HindIII fragment (35.0 to 37.7 map units). These mRNAs directed the synthesis of a 25K protein which in size was identical to the 25K protein in wild-type virus-infected cells and the translation product of a 1.15-kb cRNA transcribed from a RsaI fragment (36.4 to 37.4 map units). Comparison of gel band patterns following partial proteolysis of the translation product of the 1.15 cRNA and the 25K protein from wild-type virus-infected cells revealed that the two proteins were closely related if not identical. Nucleotide sequence analysis within this EcoRI-HindIII fragment revealed an open reading frame which encodes a 25K protein. Insertion of the Escherichia coli lacZ gene encoding the beta-galactosidase enzyme into the transcribed portion of this EcoRI-HindIII fragment yielded a recombinant virus which lacked a 25K protein and exhibited an altered (FP) plaque phenotype.

Animals↗