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G F Rohrmann

Publications and source records attributed to G F Rohrmann.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of the interaction between the baculovirus replication factors LEF-1 and LEF-2.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.

Animals↗
PubMed1997

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution.

LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein. In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis. These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer. This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry.

DNA-Binding Proteins↗
PubMed1997

A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with BglII sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (pIe-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1-specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.

Animals↗
PubMed1996

Structure-function analysis of the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region palindromes.

Homologous regions (hrs), which are present at eight dispersed locations on the Autographa californica multinucleocapsid nuclear polyhedrosis virus genome, are composed of repeated imperfect palindromes within directly repeated sequences. Hrs act as transcriptional enhancers of RNA polymerase II-mediated transcription and as origins of DNA replication when incorporated into plasmids and tested in transient replication assays. To characterize the physical structure of these elements and to determine the role that mismatched nucleotides may play in hr function, oligonucleotides containing a consensus mismatched "imperfect" palindrome and a closely related "perfect" palindrome were synthesized. These sequences were cloned into individual plasmids and tested for their ability to form cruciform structures using nuclease P1 assays and two-dimensional (2-D) gel analyses of topoisomers. The perfect palindrome formed a cruciform structure and the energy requirement for its formation was predicted to occur under physiological conditions. In contrast, the construct containing an imperfect palindrome did not form a cruciform under these conditions. Both hr constructs were found to bind IE-1 in electrophoresis mobility shift assays and act as enhancers when cis-linked to the baculovirus 39K early gene promoter. However, a single oligonucleotide containing the palindrome sequence did not bind IE-1 when annealed under conditions conducive to hairpin formation.

Animals↗
PubMed1996

Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro.

The virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells was separated from the three host nuclear RNA polymerases by DEAE-Sephadex chromatography and then purified through two more steps: heparin-agarose chromatography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayed in vitro for the ability to perform accurate initiation of transcription on a late (p6.9) and a very late (polyhedrin) template using primer extension analysis. In each case, the ability to accurately initiate transcription of these genes coincided with the virus-induced polymerase activity. Only after the glycerol gradient ultracentrifugation step did significant amounts of nonspecific late initiation occur, but specific late initiation was still readily detectable, suggesting that there is a limited number of late transcription factors, or that the factors are stably bound in a complex. After the glycerol gradient ultracentrifugation step, SDS-PAGE showed fewer than 10 prominent polypeptides remaining in the active fractions, which suggests a high degree of purity of the transcription machinery.

Animals↗
PubMed1996

The DNA polymerase and helicase genes of a baculovirus of Orgyia pseudosugata.

Regions of the genome of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) containing the DNA polymerase and helicase genes were sequenced. The DNA polymerase and helicase genes encode predicted proteins of 985 (112.6 kDa) and 1223 (140.5 kDa) amino acids and exhibited 63 % and 59 % amino acid identity, respectively, with their homologues in the Autographa californica MNPV (AcMNPV). The influence of sequence variation between the OpMNPV and AcMNPV DNA polymerase and helicase was investigated by employing gene substitution experiments in transient replication assays in Lymantria dispar and Spodoptera frugiperda cells. The DNA polymerase gene appeared to be interchangeable in this assay; both the AcMNPV and OpMNPV DNA polymerase supported high levels of replication of an origin-containing reporter plasmid when substituted for their homologue and cotransfected with a set of heterologous essential and stimulatory replication genes into uninfected insect cells. However, the OpMNPV helicase failed to support replication when it replaced the AcMNPV helicase from the set of AcMNPV replication genes cotransfected into S. frugiperda cells. In contrast, the AcMNPV helicase gene supported about 50% of the level of replication when substituted for its homologue in the OpMNPV set of replication genes.

Amino Acid Sequence↗
PubMed1995

Replication of Orgyia pseudotsugata baculovirus DNA: lef-2 and ie-1 are essential and ie-2, p34, and Op-iap are stimulatory genes.

A transient DNA replication assay was used to identify genes located within m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when cotransfected into uninfected Lymantria dispar cells. The viral transactivator ie-1 and a 2.4-kb subclone were found to be essential for replication. The 2.4-kb region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 2 (lef-2), as the essential replication gene. The OpMNPV lef-2 gene encodes a protein with a predicted molecular weight of 22.7 kDa (204 amino acids) and exhibits 54.7% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that OpMNPV lef-2 was expressed at both early and late times postinfection as a transcript of about 1.6 kb. The early transcript initiated approximately 30 nt downstream of a TAATA box, whereas the late transcript initiated from within a late promoter consensus motif. In addition, we identified three genes stimulatory for DNA replication including two OpMNPV transcriptional activators (ie-2 and p34) and Op-iap, which is the functional analog of the AcMNPV p35 gene that inhibits apoptosis in AcMNPV-infected Spodoptera frugiperda cells.

Amino Acid Sequence↗
PubMed1995

Identification, sequence, and transcriptional analysis of lef-3, a gene essential for Orgyia pseudotsugata baculovirus DNA replication.

Using a transient complementation assay for DNA replication of an origin-containing reporter plasmid, a 2.4-kb region (m.u. 48.3-50.1) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was identified that contains a gene essential for OpMNPV DNA replication. This region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 3 (lef-3), as the essential replication gene. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that lef-3 was expressed as an early gene with a major transcript of 1.7 kb in length. OpMNPV lef-3 encodes a protein with a predicted molecular weight of 42.6 kDa (373 amino acids) and exhibits 41% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. A pattern of amino acids similar to that found in single-stranded DNA binding proteins is present in the amino-terminus of both OpMNPV and AcMNPV LEF-3.

Amino Acid Sequence↗
PubMed1995

The Autographa californica nuclear polyhedrosis virus homologous region 1a: identical sequences are essential for DNA replication activity and transcriptional enhancer function.

Hr1a consists of two 30-bp imperfect palindrome sequences separated by 58 bp and each palindrome contains a naturally occurring EcoRI site at its core. Plasmid subclones of the hr1a-containing AcMNPV HindIII-N fragment were examined for their ability to replicate in virus-infected (Spodoptera frugiperda) Sf9 cells, and to stimulate transcription when linked in cis with a 39K gene promoter-beta-glucuronidase fusion and cotransfected into cells along with a plasmid (ple-1) containing the gene encoding the trans-acting factor IE-1. Only those plasmids containing hr1a underwent infection-dependent replication and were able to stimulate transcription. Sequences mapping to the left of hr1a were required for maximal levels of replication. A single palindrome from hr1a was sufficient for supporting plasmid replication and for stimulating transcription, although both activities were more efficient when both palindromes were present. Plasmids containing only one-half of a palindrome or disruptions of the central EcoRI core either did not replicate, or replicated very poorly, and did not exhibit enhanced transcriptional activity from the 39K gene promoter. Gel retardation experiments showed that labeled hr1a-containing DNA fragments had retarded migration after incubation with extracts from cells transfected with ple-1. Supershift experiments using polyclonal antibodies to IE-1 indicated that IE-1 is a component of the protein complex bound to hr1a. Fragments containing disruptions of the EcoRI-core still bound IE-1, as shown by gel retardation assays, indicating that IE-1 binding alone is not sufficient to allow replication and transcriptional enhancement.

Animals↗
PubMed1995

Identification of essential trans-acting regions required for DNA replication of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus: lef-1 is an essential replication gene.

A transient replication assay for the identification of baculovirus genes that are essential for replication of an origin-containing reporter plasmid was established for the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV). Using a replication origin located on the OpMNPV HindIII-N fragment, we identified a subset of cosmids and plasmids from an OpMNPV cosmid library that was able to supply all the essential trans-acting factors and support replication of the origin-containing plasmid in uninfected Lymantria dispar cells. However, this limited set of DNA's was unable to support replication of a second origin-containing plasmid derived from a different region of the OpMNPV genome. Replication analysis of deletion clones of the HindIII-N fragment led to the identification of a gene, late expression factor 1 (lef-1), that is essential for the transactivation of DNA replication in this system. Transcriptional analysis of lef-1 mapped both early and late transcripts of about 1.75 kb. A motif characteristic of nucleoside triphosphate-binding sites present in the carboxy-terminal region of AcMNPV Lef-1 is not conserved in OpMNPV Lef-1.

Amino Acid Sequence↗
PubMed1995

Identification and characterization of a second putative origin of DNA replication in a baculovirus of Orgyia pseudotsugata.

A 7.5-kb region (96.8-2.5 m.u.), called Op5, of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that contains an origin of DNA replication was characterized. This region replicates several times more efficiently and is unrelated to the previously identified putative origin of replication located on the viral HindIII-N fragment. In contrast to HindIII-N, the origin on Op5 contains repeated sequences with limited sequence identity to the homologous regions from AcMNPV. In isolation, these repeated sequences were not sufficient for origin activity in OpMNPV-infected Lymantria dispar cells, but required an additional 1.3 kb of sequences located to the left of the repeats. Four regions of the OpMNPV genome that crosshybridize with the repeated region were also found to replicate in our infection-dependent DNA replication assay. A deletion clone of Op5 that replicates efficiently in OpMNPV-infected L. dispar cells, was found to replicate at less than 2% the replication level of AcMNPV hr2 in AcMNPV-infected Spodoptera frugiperda cells.

Animals↗
PubMed1995

Lymantria dispar nuclear polyhedrosis virus homologous regions: characterization of their ability to function as replication origins.

Homologous regions (hrs) were identified in the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) genome. A 1.58-kb region surrounding hr4 was sequenced and found to have two distinct domains. Domain I (about 600 bp) is composed of seven repeats of about 80 bp including a series of palindromes containing MluI sites and overlapping XhoI and SacI sites. Domain II (about 700 bp) is composed of eight partially repeated sequences of 60 to 100 bp containing a 15- to 25-bp sequence that is 80 to 100% A+T in addition to a 6- to 10-bp palindrome containing an NruI site. Hybridization of a domain I sequence to cosmids containing the LdMNPV genome indicated its presence at eight positions (hr1 to -8) on the genome. In contrast, hybridization of domain II indicated that it was present only at the hr4 locus. A DpnI-based transient-replication assay was used to determine if subclones of hr4 transfected into LdMNPV-infected L. dispar cells functioned as replication origins. Subclones of hr4 containing either domain I or domain II replicated at very low or moderate levels, respectively. However, when domain I and domain II were linked on the same plasmid, high levels of replication were observed. A 1.4-kb region containing hr1 was also sequenced. It lies immediately upstream of the polyhedrin gene and contains six domain I-type repeats. Four-hundred-base-pair regions of domain I repeats from hr1 and hr4 showed 89% sequence identity. Plasmids containing the hr1 domain I replicated at low levels. However, hybrid plasmids in which the AT-rich hr4 domain II was inserted adjacent to hr1 domain I replicated to high levels, indicating that the AT-rich domain II greatly enhances replication. The orientation and position of domains I and II relative to each other did not have major effects on the levels of replication.

Animals↗
PubMed1994

Identification of genes involved in DNA replication of the Autographa californica baculovirus.

By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPV pcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1.

DNA Replication↗
PubMed1994

Characterization of the Spodoptera frugiperda TATA-binding protein: nucleotide sequence and response to baculovirus infection.

A cDNA clone containing a 921 bp open-reading frame (307 amino acids; 34 kDa) homologous to the TATA-binding protein (TBP) was isolated and sequenced from a Spodoptera frugiperda cell line that is commonly used in the baculovirus expression system. Analysis of the S. frugiperda TBP (SfTBP) sequence showed that the amino-terminal portion of SfTBP diverged significantly from that of other TBP sequences including Drosophila melanogaster whereas the carboxy-terminal sequence was highly conserved. Southern blot analysis indicated that SfTBP was encoded by a single gene in the S. frugiperda genome. Northern blot analysis indicated that steady-state levels of the 1.3 kb SfTBP transcript declined by 24 h post-infection corresponding to the time of virus-induced inhibition of host-cell transcription. Corresponding western blot analysis showed that TBP protein levels remain constant up to 72 h post-infection.

Amino Acid Sequence↗
PubMed1994

Orgyia pseudotsugata baculovirus p10 and polyhedron envelope protein genes: analysis of their relative expression levels and role in polyhedron structure.

To investigate the regulation of p10 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the p10 gene, the PEP gene or both genes were disrupted with the beta-glucuronidase (GUS) or beta-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for p10 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the p10- viruses lacked p10-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the p10-GUS+ virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by p10 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both p10 and PEP are important for the proper formation of the periphery of polyhedra.

Animals↗
PubMed1993

Analysis of the role of 5' promoter elements and 3' flanking sequences on the expression of a baculovirus polyhedron envelope protein gene.

The polyhedron envelope protein gene of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) is the third in a series of five open reading frames (ORFs 1-5) oriented in the same direction. Individual mRNAs initiate at conserved late gene promoter/mRNA start site (A/GTAAG) sequences located upstream of each ORF and the mRNAs co-terminate after the fifth ORF. To examine the influence of transcription from upstream promoter elements and the presence of an extensive 3' flanking sequence on the expression of the polyhedron envelope protein gene, the region was cloned into a phagemid vector and a BamHi site was inserted downstream of the ATG by site-directed mutagenesis and used for the insertion of a chloroamphenicol acetyl transferase (CAT) reporter gene. A set of clones were constructed in which individual or combinations of the late promoter elements from OFs 1, 2, and 3 were destroyed by site-directed mutagenesis. These plasmid constructs were transfected into Lymantria dispar cells infected with OpMNPV and cell extracts were assayed for CAT activity. Inactivation of the late promoter element immediately 5' of the polyhedron envelope protein gene led to a 96% decrease in CAT expression. Destruction of the ORF 2 late promoter element, or both the ORF 1 and ORF 2 late promoter elements, or deletion of the entire region containing ORFs 1 and 2 resulted in a 17 to 35% increase in CAT expression. In contrast, inactivation of the ORF 1 promoter alone resulted in no increase in CAT expression. Deletions of 3' flanking sequences of the polyhedron envelope protein gene caused major reduction in both CAT activity and steady-state levels of CAT mRNA.

Baculoviridae↗
PubMed1993

A baculovirus encoded 16-kDa glycoprotein localizes near the nuclear membrane of infected cells.

An open reading frame (ORF 2) located upstream of the polyhedron envelope protein gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) was cloned in frame into a trpE expression vector. The fusion protein produced by this construct was used for the production of a monospecific antiserum. Western blot analysis of OpMNPV-infected Lymantria dispar cells detected a 16-kDa protein at 24 hr postinfection. The 16-kDa protein was determined to be N-glycosylated by tunicamycin treatment of infected cells. Immunofluorescence microscopy localized the 16-kDa protein to foci of intense cytoplasmic staining near the nuclear membrane. Immunoelectron microscopy indicated that the 16-kDa protein is associated with lamellar-like structures peripheral to the nuclear membrane and with envelopes of virus that have budded into the cytoplasm. The 16-kDa protein was not associated with extracellular budded or polyhedron-derived virions.

Animals↗