Orgyia Pseudotsugata Multinucleocapsid Nuclear Polyhedrosis Virus Research Articles

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.

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.

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.

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 Hind III-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 carboxyterminal region of AcMNPV Lef-1 is not conserved in OpMNPV Lef-1.

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 13 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.

Identification and Characterization of a Putative Origin of DNA Replication in the Genome of a Baculovirus Pathogenic for Orgyia pseudotsugata

A four-kilobase (kb) region (HindIII-N, map units 7.0-11.3) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was found to contain sequences that conferred upon plasmids the ability to undergo infection-dependent replication. The plasmid DNA appeared to be replicated to form high molecular weight multimers. Plasmids with deletions of up to 1.8 kb from either end of the HindIII-N region were replication competent. However, a discrete sequence, contained within the region bracketed by the deletions, capable of specifying replication was not identified. No evidence for sequence homology was found between the OpMNPV HindIII-N region and regions elsewhere in the OpMNPV genome or to putative Autographa californica MNPV (AcMNPV) replication origins. Origin-dependent plasmid replication was shown to require the presence of the OpMNPV DNA polymerase gene. The OpMNPV origin replicated poorly in AcMNPV-infected Spodoptera frugiperda cells and, conversely, a putative AcMNPV origin (hr2) replicated at low levels in OpMNPV-infected Lymantria dispar cells.

A 25-kDa Protein Is Associated with the Envelopes of Occluded Baculovirus Virions

Antiserum produced against preoccluded virions of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was used to screen an OpMNPV λgt11 expression library. One of the immunoreactive clones contained an insert that hybridized to a portion of the OpMNPV HindIII-P fragment. A 2-kb region of HindIII-P was sequenced and found to contain three open reading frames, each preceded by a late promoter element. The insert from the λgt11 clone was derived from the third open reading frame, which encodes a predicted protein of 25 kDa. The λgt11 insert was cloned into a pMALcR1 bacterial expression vector and the fusion protein was expressed, isolated, and used for antibody production. This antiserum detected a doublet of approximately 25 kDa on Western blots of a time course of OpMNPV-infected Lymantria dispar cells. The protein was first detected at low concentration by 18 hr p.i.; by 36 hr p.i., the protein concentration had increased significantly and remained at this level for the duration of the time course. Similar results were seen on Western blots of Autographa californica MNPV (AcMNPV)-infected Spodoptera frugiperda cells. No evidence of O- or N-linked glycosylation of the OpMNPV p25 was found. Immunoelectron microscopy showed that p25 was present in the nuclei of OpMNPV-infected cells and localized to the envelopes surrounding polyhedron-derived virions.

A 37-kilodalton glycoprotein from a baculovirus of Orgyia pseudotsugata is localized to cytoplasmic inclusion bodies.

The gene encoding a 37-kDa glycoprotein (gp37) of Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was located and sequenced. gp37 of OpMNPV was found to have 62 and 37% amino acid sequence identity with gp37 of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and with a protein reported to be a component of occlusion bodies from Choristoneura biennis entomopoxvirus, respectively. The mRNA start site of the OpMNPV gp37 gene was mapped within a late promoter sequence (TTAAG). A TrpE fusion protein containing 55% of the OpMNPV gp37 gene amino acid sequence was used to generate a monospecific antiserum. Western immunoblot analysis of OpMNPV-infected Lymantria dispar cells detected gp37 beginning at 24 h postinfection. Immunoelectron microscopy indicated that the protein is concentrated in cytoplasmic inclusion bodies late in infection. In contrast to gp37 of AcMNPV which was present in the matrix of occlusion bodies, OpMNPV gp37 was not observed in this location. Neither OpMNPV nor AcMNPV gp37 was associated with the polyhedron envelope.

Immunocytochemical characterization of p24, a baculovirus capsid-associated protein.

An open reading frame (ORF 1) located upstream of the polyhedron envelope protein gene in the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was cloned in-frame into a trpE bacterial expression vector. The fusion protein produced by this construct was used for the preparation of a monospecific antiserum. Western blot analysis of extracts from OpMNPV-infected Lymantria dispar cells and Autographa californica NPV (AcMNPV)-infected Spodoptera frugiperda cells detected a 24K protein late in infection. This antiserum also reacted with a 24K protein in preparations of budded and polyhedra-derived virus from OpMNPV and AcMNPV. The 24K protein was not N-glycosylated. Immunoelectron microscopy confirmed that the OpMNPV p24 is associated with nucleocapsids of budded and polyhedra-derived virions.

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 coterminate 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 ORFs 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 reductions in both CAT activity and steady-state levels of CAT mRNA.