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G F Rohrmann

Publications and source records attributed to G F Rohrmann.

At least 19 recordsLinked to original sources
PubMed1992

A synthetic early promoter from a baculovirus: roles of the TATA box and conserved start site CAGT sequence in basal levels of transcription.

Many baculovirus early genes and insect genes transcribed by RNA polymerase II have a conserved transcription start site sequence (CAGT) located downstream of a consensus TATA box. To examine the functions and interactions of these two motifs in initiating accurately positioned basal transcription, a 43-nt synthetic promoter was synthesized from the TATA box and start site sequences of the gp64 early promoter from the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The synthetic promoter initiated accurately and was also transactivated by the baculovirus transcriptional activator, IE1. To determine the roles of sequences within the 43-nt synthetic promoter, a series of linker-scanning and spacing mutations were analyzed for transcriptional activity, start site selection, and transactivation. Linker-scanning mutations were examined in vivo by transient expression and reporter gene assays. To examine transcription start site selection, promoter constructs were used for in vitro transcription in nuclear extracts from uninfected Spodoptera frugiperda (Sf9) cells. In vivo and in vitro analyses show that the TATA box, and not the start site CAGT, is the primary element controlling start site selection. Substitution of the conserved start site CAGT sequence resulted in a reduction of both reporter gene activity and in vitro transcripts, although transcripts initiated accurately. Data from linker-scanning and spacing mutations indicate that the conserved start site CAGT sequences are not required for accurate initiation but sequences at the start site play an important role in initiation efficiency.

Baculoviridae↗
PubMed1992

Characterization of the nucleotide sequence of the Lymantria dispar nuclear polyhedrosis virus DNA polymerase gene region.

The DNA polymerase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was cloned and sequenced. The predicted DNA polymerase protein (1113 amino acids, 115.9K) was found to have an amino acid identity of 48% with the corresponding gene of the Autographa californica MNPV (AcMNPV). It contains five domains associated with substrate binding, primase interaction, and pyrophosphate hydrolysis and three domains associated with 3'-->5' exonuclease activity common to other DNA polymerases. A region with a conserved TATA promoter and a CAGT mRNA start site sequence motif was identified and shown to be transcribed by RNA polymerase II, indicating that the LdMNPV DNA polymerase gene is expressed as an early gene. An open reading frame possibly expressed as a late gene, oriented in the opposite direction and overlapping the N-terminal coding region of the DNA polymerase gene was found in the LdMNPV sequence and was shown to be conserved in the same position in AcMNPV.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of the polyhedron envelope protein gene region of the Lymantria dispar nuclear polyhedrosis virus.

A 6.4 kb region from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome was sequenced and found to contain open reading frames (ORFs) homologous to the polyhedron envelope (PE) protein coding sequence, and the C-terminal half of ORF 1, which is a gene located upstream of the PE protein gene in other baculoviruses. The proteins predicted from the LdMNPV genes encoding the PE protein, and ORF 1 demonstrated 27 and 34% amino acid sequence identity, respectively, with the corresponding genes in the Autographa californica multicapsid nuclear polyhedrosis virus.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of the p39-capsid gene region of the Lymantria dispar nuclear polyhedrosis virus.

A 1.85 kb region, containing an open reading frame (ORF) homologous to the baculovirus p39-capsid gene, was sequenced from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome. Analysis of the p39-capsid gene demonstrated that it was 39% and 47% identical in amino acid sequence with the homologous genes in the Autographa californica and Orgyia pseudotsugata MNPVs, respectively. Two late promoter elements located upstream of the p39 gene in the LdMNPV genome are conserved with two other baculoviruses, whereas an ORF located downstream is not conserved.

Amino Acid Sequence↗
PubMed1992

In vitro transactivation of baculovirus early genes by nuclear extracts from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells.

Nuclear extracts, prepared from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells during a time course of infection, were analyzed for activation of early gene transcription and for late gene transcription. The templates used in the in vitro transcription assays contained promoters for baculovirus genes that have been classified as immediate early, delayed early, and late. The promoters were derived from the baculovirus 39K, p26, gp64, and DNA polymerase genes. In addition, the adenovirus major late promoter was included in these studies. We found that transcription from promoters classified as immediate early or delayed early was accurately initiated by using extracts from uninfected cells. Furthermore, transcription from all early promoters tested was found to be transactivated by nuclear extracts prepared at 4 and 8 h postinfection. However, baculovirus enhancer-dependent transcriptional activation was not observed in tests with templates containing the hr5 enhancer sequence. Transcription from baculovirus late promoters was also not observed. A decline in transcription by nuclear extracts prepared from cells late in infection was associated with the presence of DNase activity.

Adenoviridae↗
PubMed1991

In vitro transcription of baculovirus immediate early genes: accurate mRNA initiation by nuclear extracts from both insect and human cells.

The production and characterization of nuclear extracts from uninfected Spodoptera frugiperda cells, capable of accurately initiating transcription of baculovirus immediate early genes in vitro, are described. Optimal in vitro transcription was dependent on the presence of a TATA box promoter element and was abolished by alpha-amanitin. Nuclear extracts from the S. frugiperda cells primed with plasmid DNA containing the adenovirus major late promoter produced run-off transcripts of the size predicted for initiation from the adenovirus promoter. In addition, nuclear extracts prepared from a human cell line accurately initiated transcription from the promoter of the baculovirus immediate early gene encoding gp64. Primer extension analysis showed that transcripts derived from the gp64 gene promoter using both the S. frugiperda and human cell nuclear extracts initiated at the same nucleotide as transcripts produced in vivo.

Adenoviridae↗
PubMed1991

Immunoelectron microscopic examination of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus-infected Lymantria dispar cells: time course and localization of major polyhedron-associated proteins.

Immunoelectron microscopy was employed to examine the temporal expression and localization of two proteins involved in baculovirus polyhedron assembly (polyhedrin and p10) of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) in infected Lymantria dispar cells. In addition, the association of p10 with the polyhedron envelope (PE) protein was studied. The major capsid protein (p39) was also examined to investigate the association of virion structural proteins with polyhedron formation. In infected cells, p39 did not show a concentrated association with any infected-cell structures other than nucleocapsids and appeared to be randomly distributed over the nucleocapsid surface. Likewise, polyhedrin showed no major concentrations outside of developing or mature polyhedra. The p10 antibody cross-reacted with a protein associated with condensed chromosomes in uninfected cells. In infected cells, p10 is a component of the body of fibrillar structures. The PE protein has been shown to accumulate around the periphery of fibrillar structures. Cells infected with a polyhedrin-minus virus expressing the beta-galactosidase gene under the control of the polyhedrin promoter were examined to determine whether the lack of polyhedra would influence the localization of major polyhedron-associated viral proteins. High concentrations of PE protein accumulating on the periphery of fibrillar structures appeared to be the major difference from wild-type virus-infected cells. The beta-galactosidase protein appeared to be distributed throughout the nucleus and cytoplasm, in contrast with the specific localization of the viral proteins.

Animals↗
PubMed1991

Baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by IE1.

Expression of the baculovirus major envelope glycoprotein gene (gp64) is regulated by transcription from both early and late promoters. To characterize the early promoter and identify sequences involved in the regulation of gp64 early transcription, promoter-reporter gene fusions were generated from the Orygia pseudotsugata nuclear polyhedrosis virus gp64 promoter and were analyzed by transient expression in uninfected insect cells. For these analyses, 5' deletion mutations were constructed in the gp64 upstream regulatory region. Larger promoter constructs were functional in uninfected Lymantria dispar cells, indicating that transcription from the gp64 early promoter required no additional viral gene products. Deletion analysis of the gp64 upstream region revealed several regulatory regions. These included a putative negative regulatory element between -319 and -166 nucleotides (nt) and multiple positive regulatory elements between -166 and -77 nt. Deletion of the TATA box located between -77 and -62 nt resulted in the loss of transcriptional activity. Cotransfections of reporter constructs and a plasmid containing a baculovirus transcriptional transactivator gene (Autographa californica nuclear polyhedrosis virus IE1) resulted in transcriptional transactivation of all constructs containing an intact TATA box. These data demonstrate that sequences upstream of the gp64 TATA box are not essential for IE1 transactivation and that only 34 nt upstream of the early transcription start site were necessary for basal levels of transcription and for transactivation by IE1. Function of the gp64 early promoter was also examined in cell lines from Spodoptera frugiperda and Drosophila melanogaster.

Amino Acid Sequence↗
PubMed1990

Localization of the 34 kDa polyhedron envelope protein in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus.

Using immuno-electron microscopy the 34 kDa polyhedron envelope (PE) phosphoprotein (pp 34) was localized in cells infected with Autographa californica multiple-nucleocapsid nuclear polyhedrosis virus (AcMNPV). In wild-type AcMNPV-infected cells this protein was found associated with electron-dense "spacers" and the polyhedron envelopes demonstrating their structural relationship. In these cells pp 34 was also found associated with fibrillar structures present in the nucleus and cytoplasm of infected cells. However, when cells were infected with an AcMNPV mutant with an inactivated pp 34 gene, antiserum against pp 34 still localized in fibrillar structures. In cells infected with AcMNPV mutants lacking p 10, and thus devoid of fibrillar structures, pp 34 localized normally with both electron-dense "spacers" and polyhedron envelopes. These data confirm that fibrillar structures are not essential for the morphogenesis of polyhedron envelopes and demonstrate that the association of anti pp 34 serum with these structures is specific but fortuitous.

Animals↗
PubMed1990

A baculovirus polyhedron envelope protein: immunogold localization in infected cells and mature polyhedra.

A polyclonal antiserum against a trpE fusion protein containing the complete open reading frame of the polyhedron envelope (PE) protein from the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was used for immunogold staining and electron microscopic examination of polyhedra, isolated polyhedron envelopes, and infected insect cells at selected times postinfection. The antiserum specifically stained the peripheral envelope of mature polyhedra and also stained the envelope structure which remained after polyhedra were dissolved in dilute alkaline solutions. In OpMNPV-infected Lymantria dispar cells, the PE protein was detected by 48 hr postinfection (hr p.i.) but specific localization and staining of developing polyhedra were not evident. However, by 72 hr p.i. substantial and preferential staining of the periphery of developing polyhedra was evident even though a distinct polyhedron envelope was not yet observed. In addition, the periphery of fibrillar structures was stained by the PE antiserum. By 96 hr p.i., mature envelopes surrounded polyhedra and these polyhedron envelopes were stained with the PE antibody. The progression of PE protein staining during polyhedron morphogenesis indicates that the PE protein accumulates and becomes associated with developing polyhedra in the nucleus between 48 and 72 hr p.i. Very late in infection the mature polyhedron envelope forms on the polyhedron surface. The apparent affinity of the PE protein for the surface of maturing polyhedra suggests that it may be a major component of the polyhedron envelope or may form the matrix for the deposition of other components which contribute to the mature envelope. Immunogold staining and protease digestion experiments indicate that protein is an essential component of the polyhedron envelope.

Animals↗
PubMed1990

A capsid-associated protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata: genetic location, sequence, transcriptional mapping, and immunocytochemical characterization.

Two lambda gt11 clones containing overlapping DNA inserts encoding portions of a structural protein gene from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) were identified by their immunoreactivity with polyclonal antisera produced against purified polyhedra-derived virus. Sequence analysis of a 3.6-kb region of the baculovirus genome (map units 69.1-71.6) from which the lambda gt11 inserts originated revealed an open reading frame of 1872 nt (624 amino acids) encoding a predicted protein of 70.6 kDa. Northern blot, primer extension, and 3' S1 analysis of this ORF indicated that an mRNA of approximately 2100 nt was transcribed from this gene. The mRNA appears to initiate from a late promoter/mRNA start site consensus sequence GTAAG and is expressed at late times postinfection. A gene fusion containing the C-terminal 368 amino acids of the gene was constructed using a bacterial trpE expression vector. Rabbit antiserum made against the purified fusion protein reacted with a protein of 87 kDa on Western blots of infected cell extracts at 24 hr p.i. and thereafter. The p87 protein was shown to be a component of both budded and polyhedra-derived virus and purified capsids. Immunofluorescence analysis indicated that p87 is expressed late in infection and concentrated in infected cell nuclei.

Amino Acid Sequence↗
PubMed1990

Characterization of the infection cycle of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus in Lymantria dispar cells.

To characterize the infection cycle of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus in Lymantria dispar cells, the time course of DNA synthesis and polyhedron production, and the onset and rate of budded virus production were investigated at three different m.o.i. (5, 10 and 100). In addition, the time course of expression of three proteins (gp64, p39 and polyhedrin) representative of three temporal classes of baculovirus genes was also analysed using Western blot analysis. DNA synthesis began at 12 to 18 h post-infection (p.i.). The rate of budded virus (BV) production reached maximal levels at 24 to 36 h p.i. and continued at high levels indicating that BV production was not turned off late in infection. Polyhedra were first observed at 48 h p.i. The m.o.i. appeared to influence the magnitude but not timing of early events in the viral infection cycle (gp64 expression and DNA synthesis) and also influenced the initial levels of BV production and the percentage of cells containing occlusion bodies. The m.o.i. had little influence on the final rates of BV production and the time of detection of p39 and polyhedrin on Western blots.

Animals↗
PubMed1990

The p6.5 gene region of a nuclear polyhedrosis virus of Orgyia pseudotsugata: DNA sequence and transcriptional analysis of four late genes.

The gene encoding the basic DNA-binding protein (p6.5) of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was localized by Southern blot analysis using a cDNA probe containing the Autographa californica virus (AcMNPV) p6.9 gene. The OpMNPV p6.5 gene was mapped to the HindIII G fragment at map unit 67. Nucleotide sequence and transcriptional analysis of a 3.26 kb region encompassing this area revealed four open reading frames (ORFs 1 to 4) oriented in the same direction. ORF 1 demonstrated a seven codon overlap with ORF 2. Messenger RNAs initiated upstream of each of the four ORFs late in infection and were coterminal at a single site downstream of the fourth ORF. The conserved late gene promoter/mRNA start site sequence (ATAAG) was present upstream of all the ORFs, but did not appear to be the major site of mRNA initiation for the third ORF, as determined by primer extension analysis. The fourth ORF in this series encoded a predicted peptide of 51 amino acids (6.5K), which was 80% similar to the p6.9 basic DNA-binding protein of AcMNPV.

Amino Acid Sequence↗
PubMed1990

Baculovirus diversity and molecular biology.

With the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. Since less than 30% of the AcMNPV genome has been sequenced and characterized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our understanding of the baculovirus genome and its replication. The size and diversity of baculovirus genomes appears to be strongly influenced by mobile DNA from the insect host. Also, transposon-mediated mutations of baculoviruses provide examples of functional inactivation of viral genes (FP phenotype mutations) and transcriptional activation (TE-D insertion). Another role transposable elements may play is the introduction of insect promoters and enhancers to the baculovirus genome. Since early baculovirus genes are likely transcribed in a way similar to normal insect genes, transposons that insert strong constitutive promoters or cellular enhancers near early baculovirus genes may cause mutations that are subsequently selected for. If this does occur, baculovirus early gene promoters may exhibit a great deal of variability in sequence and may resemble host promoters. Given the overall similarity between the genomes of OpMNPV and AcMNPV and the apparent absence of a region, similar to the AcMNPV HindIII-K/EcoR1-S in OpMNPV, it is intriguing to speculate that this region which contains two ORFs and the hr5 enhancer, may have been inserted into the AcMNPV genome by transposition, possibly delivering several helpful genes (35k and 94k) and a powerful enhancer. The highly repeated enhancer may have been subsequently amplified by recombination. In such a model, the acquisition of general or species-specific enhancers might influence both virulence and host range. Acquisition of general enhancers could increase the level of early gene expression, thus accelerating the cellular infection cycle and making the virus more virulent. Similarly, the acquisition of species-specific enhancers might affect host range by accelerating the infection cycle, but only in a specific host or cell type. One might therefore postulate that diversity in baculoviruses may reflect not only different selection pressures but also the diversity of mobile DNA within host insect species. Although our understanding of baculovirus diversity and molecular biology is rapidly advancing, many of the fundamental characteristics that define the unique nature of baculoviruses remain poorly understood. One fundamental feature of baculoviruses is the production of the two virion phenotypes, PDV and BV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Mapping unprocessed epitopes using deletion mutagenesis of gene fusions.

To locate the antigenic determinant recognized by a monoclonal antibody directed against a baculovirus capsid protein, a series of overlapping deletions of a fusion protein were immunologically screened with the monoclonal antibody. The immunoreactive fusion protein was derived from a restriction fragment which contained a large portion of a baculovirus capsid protein open reading frame fused in-frame with a truncated trpE gene in a bacterial (pATH3) expression system. To map the epitope, nested sets of 5' and 3' deletion mutants were generated. Mutants were characterized by the DNA insert size or by the size of the expressed fusion protein. Selected N- and C-termini truncated fusion proteins were Western blotted and incubated with the monoclonal antibody to identify mutants which retained the epitope. Plasmid DNA from mutants which flank the 5' and 3' junction of the antigenic determinant were sequenced to determine the epitope junction. By screening forty 3' deletions and sixty-four 5' deletions, the antigenic determinant was localized to a region of seven amino acids.

Amino Acid Sequence↗
PubMed1989

A baculovirus polyhedral envelope-associated protein: genetic location, nucleotide sequence, and immunocytochemical characterization.

Using a polyclonal mouse antiserum produced against purified virions of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV), two immunoreactive lambda gtII clones were identified which contained nonoverlapping insert DNAs which mapped to a single open reading frame (ORF) in the HindIII-M fragment. Analysis of nucleotide sequence data indicates that this ORF encodes a protein with a MW of 32.4 kDa. A trpE-p32 gene fusion containing the entire p32 ORF was constructed, and the fusion protein was purified and used to immunize rabbits. Western blot analysis and immunofluorescence studies using the anti-TrpE-p32 antiserum detected a polyhedra-derived virus (PDV)-associated protein of 32 kDa at 24 hr postinfection (hr p.i.). The protein was observed in the cytoplasm and nucleus at 24 hr p.i. and became concentrated in the cytoplasm late in infection. Western blot analysis and immunofluorescent microscopy of polyhedra solubilized under various conditions indicated that p32 is associated with the polyhedral envelope. The predicted amino acid sequence for p32 showed 58% amino acid identity with the predicted amino acid sequence for an ORF (ORF 3) in a similar region of the genome of the MNPV of Autographa californica (AcMNPV). The solubility properties of the p32 protein and reciprocal immunoblotting experiments indicate the OpMNPV p32 gene encodes a protein which is homologous to the polyhedral envelope-associated phosphoprotein of AcMNPV, pp34, recently reported by M.A. Whitt and J.S. Manning [(1988) Virology 163, 33-42].

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata.

The gene encoding the 39-kDa major structural protein (p39) of Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) was sequenced and transcriptionally mapped, and its expression was examined at various times postinfection. By Northern hybridization, primer extension, and S1 nuclease analysis, we identified p39 mRNAs of approximately 2600 nt. By primer extension analysis, we identified two major sets of transcripts which initiated around -48 and -96 nt upstream of the translation start codon. The transcription start sites were located within the conserved baculovirus late gene consensus sequence, ATAAG, which is duplicated in the p39 5' flanking region. In OpMNPV-infected Lymantria dispar cells, the p39 mRNAs were expressed abundantly at 24 and 36 hr p.i. but were present in lower quantities at 48 hr p.i. The p39 gene contained an open reading frame of 1053 nt which encodes a predicted protein of 351 amino acids with an estimated molecular weight of 39.5 kDa. Three repeats of the amino acid sequence Ala-Pro-Ala-Ala-Pro were identified at the C-terminus of the predicted p39 protein.

Amino Acid Sequence↗