Orgyia Pseudotsugata Multicapsid Nuclear Polyhedrosis Virus Research Articles

Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp

Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively . The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.

Identification, sequence analysis, and phylogeny of the immediate early gene 1 of the Trichoplusia ni single nucleocapsid polyhedrosis virus.

Substantial research has been conducted on the immediate early I (ie-1) genes from the prototype baculovirus Auographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). In both cases ie-1 gene products have been implicated in transcriptional activation and repression. In this study an ie-1 homolog was identified from Trichoplusia ni single nucleocapsid polyhedrosis virus (TniSNPV). Nucleotide sequence analysis indicated that the TniSNPV ie-1 gene consists of a 2,217 nucleotide open reading frame (ORF), encoding a protein with a molecular mass of 84.464 kDa. This represents the largest baculovirus ie-1 gene characterised to date. Of the seven ie-1 homologs identified to date, the TniSNPV ie-1 shared most sequence similarity with the ie-1 gene of Spodoptera exigua MNPV (SeMNPV) (41%). At the nucleotide level, expected TATA and CAGT motifs were found to precede each ie-1 ORE. At the protein level, it was confirmed that the N-termini are poorly conserved, but share the characteristic of having a high proportion of acidic amino acids. In addition it was found that N-terminal regions significantly matched the SET domain in the Swiss-Prot prosite database. The C-terminal regions of the deduced IE-1 sequences were found to be substantially more conserved than the N-termini. Several conserved motifs were identified in the C-terminal sequences. A phylogenetic tree of nine baculovirus IE-1 proteins was constructed using maximum parsimony analysis. The phylogenetic estimation of the ie-1 genes shows that TniSNPV is a member of the previously described lepidopteran NPV group II and it is most closely related to SeMNPV.

The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection.

To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (SfpOP64-6) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64 EFP-null virus (vAc64z) contained OpMNPV GP64 EFP supplied by the Sf9OP64-6 cells. Virions propagated in Sf9OP64-6 cells were capable of infecting wild-type Sf9 cells, and cells infected by vAc64z exhibited a blue phenotype in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Using cytochemical staining to detect vAc64z infected cells, we demonstrated that this GP64 EFP-null virus is defective in cell-to-cell propagation in cell culture. Although defective in cell-to-cell propagation, vAc64z produces occlusion bodies and infectious occlusion-derived virions within the nucleus. Occlusion bodies collected from cells infected by vAc64z were infectious to midgut epithelial cells of Trichoplusia ni larvae. However, in contrast to infection by a control virus, infection by vAc64z did not proceed into the hemocoel. Analysis of vAc64z occlusion bodies in a standard neonate droplet feeding assay showed no virus-induced mortality, indicating that occluded virions produced from vAc64z could not initiate a productive (lethal) infection in neonate larvae. Thus, GP64 EFP is an essential virion structural protein that is required for propagation of the budded virus from cell to cell and for systemic infection of the host insect.

The Baculovirus GP64 Envelope Fusion Protein: Synthesis, Oligomerization, and Processing

The baculovirus GP64 envelope fusion protein (GP64 EFP) is a class I integral membrane protein that enters the secretory pathway and is oligomerized and extensively processed during transport to the plasma membrane. The kinetics of GP64 EFP biosynthesis, oligomerization, and processing in Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV)-infected Lymantria dispar cells were examined by pulse label, pulse-chase, and immunoprecipitation experiments. Relative rates of GP64 EFP synthesis in OpMNPV-infected L. dispar cells were examined at various times throughout the infection cycle. Using pulse labeling and immunoprecipitation, GP64 EFP synthesis was detected within 2 hr p.i., and the maximal rate of synthesis was observed in the period of 24-26 hr pi., a time coincident with the onset of high level production of budded virus in OpMNPV-infected L. dispar cells. To determine the oligomeric structure of GP64 EFP, a soluble form of OpMNPV GP64 EFP was produced and examined by a combination of gel filtration chromatography, nonreducing SDS-PAGE, and mass spectrometry. Oligomeric GP64 EFP was identified as a trimeric molecule, that migrates as two discrete bands on nonreducing SDS-PAGE. Pulse-chase studies, performed at both early (12 hr pi.) and late (36 hr p.i.) stages of the infection cycle, showed that GP64 EFP oligomerization is complete within 15 min after synthesis. Efficiency of oligomerization however was relatively low, with less than 33% of the synthesized GP64 EFP converted to trimers. The majority of monomeric GP64 EFP remaining in the cell appeared to be degraded within 30 to 45 min after synthesis. Analysis of the kinetics of carbohydrate processing at early (12 hr p.i.) and late (36 hr p.i.) times postinfection showed that for beth early and late phases of infection, carbohydrate was rapidly added, and processing began between 10 and 20 min after GP64 EFP synthesis. Although carbohydrate processing was completed within approximately 90 min after synthesis during the early phase, the same process required approximately 150 min during the late phase. Thus, carbohydrate processing appeared to become less efficient as infection progressed. These studies thus show that GP64 EFP undergoes a rapid but inefficient oligomerization step that results in a homotrimeric structure for GP64 EFP. While carbohydrate addition is rapid, carbohydrate processing requires prolonged periods of time (with half-times of 45 to 75 min) and appears to become less efficient during the late phase of the infection.

Analysis of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus trans-activators IE-1 and IE-2 using monoclonal antibodies.

We have produced monoclonal antibodies against two Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) transcriptional trans-activators, IE-1 and IE-2. Temporal analysis of IE-1 and IE-2 proteins have shown that IE-1 continues to increase in steady-state levels from 0 to 120 h post-infection, whereas IE-2 declines by 36 h post-infection. At least five different electrophoretic forms of IE-1 are present in OpMNPV-infected LD652Y cells of which three appear to be from the non-spliced IE-1 gene. Cotransfection experiments also showed that IE-1 causes a reduction in the levels of IE-2 protein but concurrently IE-2 increases IE-1 expression. Western blot analysis indicated that a form of IE-1 copurified with budded virions but did not copurify with the polyhedron-derived virus.

Characterization of an early gene coding for a highly basic 8.9K protein from the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus

A new gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) has been identified that encodes a highly basic 8.9K protein. The gene called p8.9 is expressed as a 0.5 kb transcript by 1 h post-infection and initiates at an early gene motif. The promoter of the 0.5 kb transcript has two upstream elements, repeats I and II, which are similar to motifs previously characterized in the OpMNPV IE-2 gene and the Autographa californica nuclear polyhedrosis virus IE-N and PE38 genes. A second p8.9 transcript expressed from 8 to 72 h post-infection was shown to initiate 634 bp upstream from the early gene motif in a region that has no similarity to any previously described baculovirus promoter or initiation site. Transient assays utilizing a reporter gene have shown that the p8.9 early promoter is active in a Lymantria dispar (LD652Y) and Spodoptera frugipeda (Sf9) cell lines in the absence of other viral factors. In addition, it was also demonstrated that the p8.9 promoter is trans-activated by the OpMNPV IE-2 and p34 genes, but not by IE-1.

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.

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 prompter 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.

Tandemly repeated sequence at the 3′ end of the IE-2 gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus is an enhancer element

An enhancer element was identified in the virus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) that is located adjacent to the 3' end of the IE-2 gene and 5′ to an open reading frame that codes for a predicted protein that has 37% homology to the AcMNPV PE-38 gene. The OpMNPV enhancer (OpE) consists of a 66-bp element that is tandemly repeated partially or completely 12 times. The OpE sequences were shown to increase gene expression from the Autographa californica MNPV delayed early p39 promoter independently of position or orientation, and were also shown to increase expression from the promoter of the OpMNPV immediate early gene, IE-2. Sequences homologous to the OpE sequences were mapped by Southern blot hybridization to four additional locations around the OpMNPV genome. This indicates that OpMNPV is similar to other baculoviruses such as AcMNPV, Lymantria dispar MNPV, and Choristoneura fumiferana MNPV that have homologous regions in several locations in the genome.

Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus

A second immediate early (IE) regulatory gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) has been identified. The IE-2 gene which is homologous to the IE-N gene of Autographa californica MNPV was mapped to the HindIII A fragment of OpMNPV between 0.41 to 1.37 map units. The IE-2 gene codes for a predicted protein of 45,640 Da and analysis of the amino acid sequence shows that the protein has a highly basic amino terminal domain and a cysteine-rich domain that is similar to a zinc finger motif that is also found in the baculovirus proteins GC30 and PE-38. The I E-2 gene is expressed as a 1.3-kb transcript that was detectable by 0.5 hr postinfection (hr p.i.), reached maximum steady state levels by 6 hr p.i., and declined slightly by 48 hr p.i. Cis-acting 5′ regulatory sequences were analyzed by deletion analysis of the IE-2 promoter linked to the reporter gene chloramphenicol acetyl transferase. Maximum expression was obtained when the IE-2 promoter contained sequences 275 by upstream from the transcriptional start site, trans-Activation analysis revealed that IE-2 trans-activated the IE-1 promoter and in addition appeared to be autoregulatory.

Identification and characterization of the ie-1 gene of orgyia pseudotsugata multicapsid nuclear polyhedrosis virus

The IE-1 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) was mapped between 95.7 and 97.1 map units on the viral genome. Sequence analysis of the OpMNPV IE-1 gene (OpIE-1) identified an open reading frame that coded for a predicted protein of 560 amino acids with a molecular weight of 64,775. Transcriptional analysis of OpMNPV-infected Lymantria dispar (LD652Y) cells identified two RNAs homologous to the OpIE-1 open reading frame that were 1.7 and 1.9 kb in size. The 1.7-kb transcript could be detected by 0 hr postinfection (hr p.i.) and the steady-state levels increased up to 48 hr p.i. The 1.9-kb message appears to be spliced and has peak expression from 4 to 6 hr p.i. but can still be detected at late times p.i.. Comparison of the OpIE-1 and Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) IE-1-predicted proteins revealed that the N-terminal region had very low sequence identity (21%) but had maintained an acidic profile, whereas the C-terminal region showed 55% amino acid identity. Transient assay analysis showed that OpIE-1 was able to trans-activate the AcMNPV delayed early reporter gene construct p39CAT in both LD652Y cells and Spodoptera frugiperda (Sf9) cells. The expression of p39CAT trans-activated by OpIE-1 was also found to be enhanced by the AcMNPV hr enhancer sequences. The OpIE-1 promoter was linked to the chloramphenicol acetyl transferase gene and deletion analysis was used to identify regions involved in the regulation of this gene. This analysis revealed that the OpIE-1 promoter contained regions that were responsive to a transcriptional activator that was specific to Sf9 cells. In addition it was shown that OpIE-1 could trans-activate its own promoter and that for maximal expression this required sequences between −420 and −330 relative to the transcriptional start site. These data suggest that OpIE-1 is autoregulated during normal viral infection of insect cells.

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.

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

Two λgt1 1 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 λgt11 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.

Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus

The gene encoding gp64, the envelope glycoprotein of the budded virus (BV) of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV), was mapped to the HindIII-E fragment of the viral genome and expression of the gp64 gene was examined at various times postinfection. To locate the gp64 gene, a cross-reacting monoclonal antibody (AcV 5) ( A. W. Hohmann and P. Faulkner, 1983, Virology, 125,432–444) directed against the gp64 protein of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was used to screen a λ gt11 expression library of OpMNPV and insert DNAs from immunopositive recombinants were used for Southern hybridization mapping. The gp64 gene was sequenced and transcription of the gp64 gene was examined by Northern blot, S1 nuclease, and primer extension analysis. Two sets of gp64 transcripts were detected during infection: a single early transcript which initiated at −43 nt and four late transcripts which initiated at −152, −167, −174, and −175 nt relative to the start of the gp64 open reading frame. Comparison of the gp64 early transcription initiation site with several other early baculovirus genes revealed a four-nucleotide consensus sequence (CAGT) which is conserved at the early transcription initiation sites of the IE-1 and 39K genes. The four late gp64 transcripts initiated at two of the four upstream ATAAG motifs. All gp64 mRNAs appear to be coterminal at the 3′ end. Analysis of the nucleotide sequence of the gp64 gene revealed that the late gp64 mRNAs are bicistronic, consisting of a three amino acid minicistron located 70 nt upstream of the 509 amino acid gp64 open reading frame. Early transcripts do not contain the minicistron. The 1527-nt gp64 open reading frame encodes a predicted protein of 509 amino acids with a molecular weight of 58 kDa. The predicted gp64 protein contains seven potential N-linked glycosylation sites and hydrophobic N- and C-termini characteristic of signal and membrane anchor sequences found on envelope glycoproteins. By western blot analyses and indirect immunofluorescence microscopy, we show that the gp64 protein is present at early times (6 hr) postinfection and accumulates in the infected cell, moving to the periphery at later times postinfection. Western blot comparisons of the temporal expression of the gp64 protein with the p39 capsid protein revealed that these two virion structural protein genes differ significantly in the timing of their initial expression. The upstream regulatory regions, open reading frames, and predicted proteins from the OpMNPV and AcMNPV gp64 genes were compared. In the upstream regulatory region, three sequences were highly conserved: (a) sequences surrounding the upstream late promoter (ATAAG) motif, (b) sequences including a putative TATA box of an early promoter, and (c) sequences containing the early transcription initiation site. The two predicted amino acid sequences show 78% amino acid identity. Evolutionary implications of the conservation between the two genes are discussed.

Production of polyhedrin monoclonal antibodies for distinguishing two Orgyia pseudotsugata baculoviruses.

Monoclonal antibodies were produced to polyhedrins from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) and single-capsid nuclear polyhedrosis virus (OpSNPV). Although the polyhedrins are closely related, antibodies were selected which allowed differentiation between the two viruses. In an indirect enzyme-linked immunosorbent assay, purified OpMNPV and OpSNPV polyhedrins could be detected by specific monoclonal antibodies at concentrations as low as 2 and 5 ng/ml, respectively. The antibodies were also capable of identifying their homologous polyhedrin in extracts of infected insects. These antibodies would be useful for monitoring production of the viral insecticide, TM Biocontrol-1, which by license must contain only OpMNPV, and to confirm that insect mortality after aerial spraying with this insecticide is attributable to OpMNPV infection.