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D L Jarvis

Publications and source records attributed to D L Jarvis.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mapping functional domains in AcMNPV pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic stroma. The virogenic stroma is the site of viral DNA packaging and nucleocapsid assembly and is thought to be the site of viral DNA replication and RNA transcription. AcMNPV encodes a phosphoprotein, pp31, which localizes to the nucleus of uninfected insect cells and to the virogenic stroma of infected insect cells. pp31 has DNA binding activity and has been identified as a late expression factor. Thus, the intracellular location of pp31, its DNA binding activity, and its identification as a late transcription factor suggest that it participates in replicative events that occur in the virogenic stroma during AcMNPV infection. The purpose of this study was to map the pp31 domains needed for nuclear localization, virogenic stroma localization, and DNA binding. We focused on four basic amino acid regions (BRs 1-4) and used site-directed mutagenesis and gene fusion techniques to probe their functions. The amino-terminal basic region (BR1) was most important for nuclear localization of pp31 in uninfected cells. Three of the four BRs were needed to efficiently localize pp31 to the nucleus and virogenic stroma in infected cells. BR3 was identified as the DNA binding domain of pp31. These data indicated that BR1, BR3, and BR4 are important functional or multifunctional domains within the AcMNPV pp31 protein.

Animals↗
PubMed1996

Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells.

Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of the polyhedrin promoter, which provides high-level transcription during the very late phase of infection. For some applications, including foreign glycoprotein production and insect pest control, it might be advantageous to have baculovirus vectors that could express foreign gene products in uninfected cells or earlier after infection. To fulfill this need, we have constructed a new set of plasmids that can be used to clone and express foreign genes under the control of a baculovirus ie1 promoter, which is active in uninfected insect cells and throughout infection. We used a subset of these new plasmids to isolate recombinant baculoviruses containing various foreign genes and compared expression of these genes by the resulting immediate-early baculovirus vectors and by conventional baculovirus vectors. As expected, the immediate-early vectors began to express each foreign gene earlier in infection but, by 36-48 h postinfection, the conventional vectors had produced more of each foreign protein. Conventional baculovirus vectors also produced more enzymatic activity from two different procaryotic genes than the immediate-early baculovirus vectors. However, immediate-early vectors produced as much or more enzymatic activity from two different eucaryotic genes encoding secretory pathway proteins than the conventional vectors, even at 48 h postinfection. Hence, this report describes a new set of plasmids that can be used to clone and express foreign genes under the control of the baculovirus ie1 promoter and suggests that immediate-early baculovirus vectors might be as useful as conventional baculovirus expression vectors for producing biologically active eucaryotic secretory pathway proteins.

Animals↗
PubMed1996

Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.

The baculovirus-insect cell expression system is well-suited for recombinant glycoprotein production because baculovirus vectors can provide high levels of expression and insect cells can modify newly synthesized proteins in eucaryotic fashion. However, the N-glycosylation pathway of baculovirus-infected insect cells differs from the pathway found in higher eucaryotes, as indicated by the fact that glycoproteins produced in the baculovirus system typically lack complex biantennary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues. We recently developed a new type of baculovirus vector that can express foreign genes immediately after infection under the control of the viral ie1 promoter. These immediate early baculovirus expression vectors can be used to modify the insect cell N-glycosylation pathway and produce a foreign glycoprotein with more extensively processed N-linked oligosaccharides. These vectors can also be used to study the influence of the late steps in N-linked oligosaccharide processing on glycoprotein function. Further development could lead to baculovirus-insect cell expression systems that can produce recombinant glycoproteins with complex biantennary N-linked oligosaccharides structurally identical to those produced by higher eucaryotes.

Animals↗
PubMed1996

Dynamic phosphorylation of Autographa californica nuclear polyhedrosis virus pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.

DNA-Binding Proteins↗
PubMed1995

Biochemical analysis of the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells.

The baculovirus-insect cell system is used routinely for foreign glycoprotein production, but the precise nature of the N-glycosylation pathway in this system remains unclear. Some studies indicate that these cells cannot process N-linked oligosaccharides to complex forms containing outer-chain galactose and sialic acid, while others indicate that they can. In this study, we used the major virion envelope glycoprotein of the baculovirus Autographa california multicapsid nuclear polyhedrosis virus (AcMNPV) to probe the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells. The results showed that gp64 contained mannose, fucose, and probably N-acetylglucosamine, but no detectable galactose or sialic acid. These same results were observed with gp64 produced in any one of three different lepidopteran insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, or Estigmene acrea, whether it was produced at relatively earlier or later times after infection. These results indicated that the gp64 produced in AcMNPV-infected lepidopteran insect cells lacks complex N-linked oligosaccharides containing outer-chain galactose and sialic acid. By contrast, gp64 produced in mammalian cells contained both galactose and sialic acid, and endoglycosidase digestions revealed that these sugars were constituents of N-linked, not O-linked, oligosaccharides. This showed that at least one N-linked side chain on gp64 has the potential to be processed to a complex form. Together, these results suggest either that AcMNPV-infected lepidopteran insect cells are unable to convert any of the N-linked side chains on gp64 to complex structures or that outer-chain galactose and sialic acid residues are added to gp64 and then removed by cellular or viral exoglycosidases.

Acetylglucosamine↗
PubMed1994

Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein.

gp64 is a major virion envelope glycoprotein of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). gp64 plays an important role in AcMNPV infection, probably mediating penetration of one form of the virus into host cells through the endocytic pathway. gp64 also represents an excellent probe for studying the membrane glycoprotein processing capabilities of baculovirus-infected insect cells, which are used widely as a eucaryotic expression system. The goals of this study were to characterize gp64 biosynthesis and processing and determine how N-glycosylation and N-linked oligosaccharide processing influence the fate and function of gp64 in AcMNPV-infected insect cells. We found that gp64 was synthesized in a biphasic fashion, with peaks at 8 and 24 hr postinfection in both the intracellular and extracellular fractions. Interestingly, the first peak preceded detectable budded virus (BV) production, suggesting that gp64 is shed from infected cells early in infection. Transcriptional regulation accounted for the biphasic mode of gp64 protein synthesis, as transcription initiated at a consensus early motif during early times of infection, at a late motif during late times of infection, and there was a lag between the peak of early and the onset of late transcription. In vitro transcription-translation assays showed that the second ATG in the AcMNPV gp64 long open reading frame is used as the translational initiation codon and that downstream sequences encode a functional signal peptide. Pulse-chase analyses, endoglycosidases, and various inhibitors were used to show that some N-linked oligosaccharides on gp64 are processed by glucosidases and alpha-mannosidases in AcMNPV-infected insect cells. These experiments also revealed that at least two differentially processed gp64 glycoforms are produced in these cells and that both can reach the cell surface and assemble into progeny BV. However, N-linked oligosaccharide processing was not required for gp64 cell surface expression, its assembly into infectious BV, or its fusogenic activity. This suggested that any gp64 glycoform produced during infection, regardless of its N-linked carbohydrate structure, can have essentially normal biological properties. By contrast, transport of gp64 to the cell surface, production of infectious BV, and fusogenic activity were reduced in the absence of N-glycosylation, indicating that this modification is necessary for optimal gp64 function.

Amino Acid Sequence↗
PubMed1994

Long-term stability of baculoviruses stored under various conditions.

The long-term stability of baculoviruses under various storage conditions was investigated. The results showed that the titers of baculovirus working stocks were significantly reduced after the viruses had been stored in polypropylene tubes for several months or a year at 4 degrees C. The virus was not lost by sticking to the plastic tubes, because the same results were obtained when the virus was stored in glass tubes. The major factor contributing to the loss of viral infectivity was exposure to light. Virus stocks stored at temperatures ranging from -85 degrees to 37 degrees C were quite stable, as long as they were protected from light. Finally, viral infectivity was not significantly reduced by multiple freeze-thaw cycles. These results demonstrated that baculovirus working stocks may be stored frozen or refrigerated with little long-term loss of infectivity, as long as they are protected from light.

Baculoviridae↗
PubMed1993

Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system.

Foreign secretory pathway proteins are often produced in surprisingly low amounts in the baculovirus/insect cell expression system. One possible reason for this is that heterologous signal peptides might be inefficiently recognized by the insect cell protein translocation machinery. This idea was supported by a recent study showing that secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide (Tessier, D. C., Thomas, D. Y., Khouri, H. E., Laliberte, F., and Vernet, T. (1991) Gene (Amst.) 98, 177-183). We have extended these observations by measuring the effects of different signal peptide and signal peptide-prosequence combinations on baculovirus-mediated expression and secretion of human tissue plasminogen activator (t-PA). Replacement of the native prepropeptide with signal peptides from a lepidopteran insect secretory protein (cecropin B), a major baculovirus structural glycoprotein (64K), or an abundant, highly conserved lumenal protein of the rough endoplasmic reticulum (GRP78/BiP, a 78-kDa glucose-regulated protein/immunoglobulin heavy chain-binding protein), had no significant effect on t-PA expression or secretion. The same results were obtained with the signal peptide from honeybee prepromellitin, which was able to enhance secretion of plant propapain (Tessier et al., 1991 (above)). Similar results were obtained when heterologous signal peptides were combined with the native prosequence or when the intact cecropin B preprosequence was used. Translational initiation at an upstream, in-frame ATT, which could functionally inactivate any signal peptide, did not explain the low efficiency of t-PA secretion. Finally, deletion of the native signal peptide, prosequence, or both, failed to increase t-PA production. These results showed that insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. They also suggested that the inability of insect cells to recognize the processing signals in human t-PA efficiently is probably not the major factor preventing its high level production in this system.

Amino Acid Sequence↗
PubMed1993

Effects of baculovirus infection on IE1-mediated foreign gene expression in stably transformed insect cells.

Previously, we produced transformed insect cell lines that can express a selected foreign protein constitutively, in the absence of baculovirus infection (D. L. Jarvis, J. G. W. Fleming, G. R. Kovacs, M. D. Summers, and L. A. Guarino, Bio/Technology 8:950-955, 1990). These cells contain stably integrated copies of chimeric genes consisting of the promoter from an immediate-early baculovirus gene, IE1, and the sequences encoding either human tissue plasminogen activator or Escherichia coli beta-galactosidase. Transcription of the integrated genes in these cells is specifically controlled by the IE1 promoter. The purpose of this study was to determine how baculovirus infection influences IE1-mediated foreign protein production by these stably transformed insect cell lines. The results showed that viral infection transiently stimulated and then strongly inhibited the production of both tissue plasminogen activator, a secreted protein, and beta-galactosidase, an intracellular protein. These effects reflected virus-induced changes in the steady-state levels of RNA produced by the integrated genes. Transient assays showed that expression of the viral IEN gene alone could account for the increased levels of RNA observed early in infection. The precise mechanism accounting for the decreased levels of RNA observed later in infection was not determined. However, we obtained evidence that the native IE1 promoter remains active throughout infection, which suggested indirectly that the integrated IE1 promoter is transcriptionally inactivated at late times of baculovirus infection. Thus, the same promoter behaved quite differently late in infection, depending on its local environment. Neither methylation nor degradation appeared to be responsible for inactivating IE1-mediated expression of the integrated genes. The significance of these results with respect to the baculovirus-host interaction and the practical applications of stably transformed insect cell lines are discussed.

Animals↗
PubMed1992

Enhancement of polyhedrin nuclear localization during baculovirus infection.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). Since viral occlusions are responsible for the horizontal transmission of AcMNPV in nature, the biosynthesis, localization, and assembly of polyhedrin are important events in the viral replication cycle. We recently defined the sequence requirements for nuclear localization and assembly of polyhedrin. In this study, we examined the localization of polyhedrin at different times of infection. The results showed that nuclear localization of polyhedrin becomes more efficient as the occlusion phase of infection progresses. Several different factors were identified that might contribute to this overall effect, including a higher rate of polyhedrin nuclear localization and a higher rate of polyhedrin biosynthesis. We also examined the biosynthesis and processing of polyhedrin in cells infected with an AcMNPV few polyhedra (FP) mutant, which produces smaller numbers of viral occlusions that contain few or no virions. Compared with wild type, the FP mutant produced polyhedrin more slowly and localized it to the nucleus less efficiently at the beginning of the occlusion phase of infection (24 h postinfection). This supported the idea that the efficiency of polyhedrin nuclear localization is tightly coupled to its rate of biosynthesis. It also revealed that expression of the viral 25K gene, which is inactivated in the FP mutant, is directly or indirectly associated with an enhancement of polyhedrin biosynthesis and nuclear localization at the beginning of the occlusion phase of infection. This enhancement effect appears to be necessary to ensure the normal assembly of viral occlusions.

Animals↗
PubMed1992

Baculovirus phosphoprotein pp31 is associated with virogenic stroma.

The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.

Animals↗
PubMed1991

Requirements for nuclear localization and supramolecular assembly of a baculovirus polyhedrin protein.

This study defines the requirements for the nuclear localization, stable nuclear association, and supramolecular assembly of a baculovirus polyhedrin protein in lepidopteran insect cells. Fragments of the polyhedrin protein were genetically fused to two different nonnuclear reporter proteins and the intracellular distribution of the fusion proteins was analyzed in infected insect cells. Analysis by indirect immunofluorescence showed that the domain between amino acids 30 and 57 could mediate nuclear localization of polyhedrin. However, biochemical fractionation experiments showed that this domain was not sufficient for a detergent-stable association of polyhedrin with the nucleus. This required a slightly larger domain, between amino acids 30 and 110. Differential interference-contrast microscopy showed that the supramolecular assembly of polyhedrin into nuclear occlusion-like particles required the domain between amino acids 19 and 110. The most likely candidate for a minimal nuclear localization signal was the sequence KRKK, located between amino acids 32 and 35. Therefore, oligonucleotide-directed mutagenesis was used to change this sequence to NGNN and the intracellular distribution of the mutant protein was analyzed. The results showed that the mutant protein was predominantly localized in the cytoplasm of infected cells, where it assembled into large, cubic, occlusion-like particles. Thus, the KRKK sequence is necessary for the nuclear localization of polyhedrin, but nuclear localization is not required for its supramolecular assembly into occlusion-like particles.

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

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

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

SV40 T-antigen as a dual oncogene: structure and function of the plasma membrane-associated population.

SV40 T-antigen (T-ag) is localized in both the nucleus (nT-ag) and plasma membrane (pmT-ag) of cells and provides multiple functions necessary for cell transformation. The pmT-ag population is structurally very similar to the nT-ag. Transport to the cell surface is by an unknown mechanism that does not involve the secretory pathway. The disposition of T-ag in the membrane exposes both the amino and the carboxyl terminus on the exterior of the cell. Nuclear-transport-defective mutants of T-ag can transform established cells in culture, but not primary cells, suggesting that non-nuclear forms of T-ag may mediate some transformation-related process(es). A non-cytolytic protein extraction technique utilizing 1-butanol solubilized from SV40-transformed cells a multimeric complex composed of pmT-ag and at least five cellular proteins ranging in size from 35,000 (35K) to 60K M. Both amino- and carboxylterminal T-ag-specific monoclonal antibodies co-precipitated T-ag and the 35-60K Mr proteins, but antibodies against the internal portion of T-ag precipitated only uncomplexed T-ag. The growth state of the cells markedly influenced the expression of the T-ag-containing surface complexes; more complexes were recovered from actively dividing cells than from confluent cell cultures, and suspension cells yielded more complexes than cells on a substratum. The complex exhibited a highly dynamic association with the cell membrane, as demonstrated by pulse-chase analysis. The characteristics of growth-dependent expression and rapid turnover rate suggest a functional role for the membrane complex. The identities of the cellular proteins in the complex with pmT-ag are unknown, although one member (56K) is recognized by p53-specific monoclonal antibodies.

Animals↗