Orgyia Pseudotsugata Multicapsid Nuclear Polyhedrosis 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.

Overlapping TATA-dependent and TATA-independent early promoter activities in the baculovirus gp64 envelope fusion protein gene

In previous studies to characterize basal and activated transcription from the early promoter of the gp64 envelope fusion protein (efp) gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus, the TATA box was identified as a functional element, essential for basal transcription from a minimal promoter construct. In the current study, we used discrete deletions and multiple point mutations that removed the functional TATA box from larger promoter constructs of the gp64 efp gene to reveal an overlapping TATA-independent transcriptional activity. TATA-independent transcriptional activity was inhibited in vitro by alpha-amanitin but not by tagetitoxin, demonstrating that like the TATA-dependent activity, the TATA-independent activity is mediated by RNA polymerase II. Using constructs in which the TATA box (TATATAA) was destroyed by substitution mutations, we identified four elements that are required for the TATA-independent activity. Two of the required elements, GATA (at -114) and CACGTG (at -104), were previously shown to specifically bind host transcription factors and activate transcription from the TATA-dependent wild-type gp64 efp promoter. The role of the early start site consensus CAGT sequence in TATA-independent transcription was also examined. Single-nucleotide substitution mutations in the CAGT sequence indicated that certain nucleotides within the CAGT start site were essential. In addition to the start site sequence and two upstream elements, a fourth essential element was identified in the 5' untranslated leader region (5'UTR). While the 5'UTR was not necessary for TATA-dependent transcription, deletion of a 10-bp 5'UTR sequence resulted in the loss of TATA-independent transcriptional activity. Although necessary, neither the GATA, CACGTG, start site region, nor 5'UTR element was alone sufficient for accurately initiated TATA-independent transcription from the consensus CAGT start site. Thus, the gp64 efp early promoter contains overlapping TATA-dependent and TATA-independent transcriptional activities. A number of common sequence elements (GATA, CACGTG, and start site CAGT) are involved in both of these activities, while one element (in the 5'UTR) is required only in the TATA-independent context.

A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements

The early promoter of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 gene is active when transfected into several insect cell lines and does not require viral gene products for transcription in uninfected cells. Because previous studies have shown that the gp64 early promoter is activated above basal levels in uninfected cells, host transcription factors are likely to play a role in gp64 activation at early times postinfection. By using nuclear extracts from uninfected Sf9 cells for electrophoretic mobility shift analysis of gp64 regulatory regions, host nuclear proteins were shown to bind specifically to the upstream regulatory region of the gp64 early promoter. Host factor binding was mapped to a 24-bp sequence centered approximately 35 bp upstream of the TATA box. Two consensus eukaryotic transcription factor-binding site motifs, GATA and CACGTG, were identified within the 24-bp sequence. Competition assays using oligonucleotides containing either a GATA or a CACGTG motif and similar oligonucleotides with point mutations in these sites showed that each site is required for binding host transcription factors. To investigate the functional significance of host factor binding to GATA and CACGTG motifs, constructs containing point mutations in these motifs were examined in transient expression assays. Mutations in either or both GATA and CACGTG sites decreased reporter activity in transient expression assays, suggesting that binding of host transcription factors to these motifs is important in transcriptional regulation of the gp64 early promoter.

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.

Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion

The baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus (BV) and is involved in BV entry into the host cell by endocytosis. To determine whether gp64 alone was sufficient to mediate membrane fusion, the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 protein was transiently expressed in uninfected insect cells. Cells expressing the baculovirus gp64 protein were examined for membrane fusion activity by using a syncytium formation assay under various conditions of exposure to low pH. Cells expressing the gp64 protein mediated membrane fusion and syncytium formation in a pH-dependent manner. A pH of 5.5 or lower was required to induce membrane fusion. In addition, exposure of gp64-expressing cells to low pH for as little as 5 s was sufficient to induce gp64-mediated syncytium formation. These studies provide direct evidence that gp64 is a pH-dependent membrane fusion protein and suggest that gp64 is the protein responsible for fusion of the virion envelope with the endosome membrane during BV entry into the host cell by endocytosis.

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.

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.

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.

Nucleotide sequencing and transcriptional mapping of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus p10 gene

A 32P-labeled cloned DNA fragment (AcMNPV HindIII-Q) containing one of the repeated sequences and a portion of the p10 gene from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was used to probe Southern blots containing restriction endonuclease digests of Orgyia pseudotsugata muticapsid nuclear polyhedrosis virus (OpMNPV) DNA. A single 3.6-kb fragment, OpMNPV HindIII-Q, was hybridized. The OpMNPV HindIII-Q fragment was cloned into pUC-18, mapped with restriction endonucleases, and reprobed with the AcMNPV HindIII-Q fragment. A small region of ca. 700 bp, near the left end of the cloned fragment, was cross-hybridized. DNA sequencing in this region revealed an open reading frame of 279 by which shares detectable homology with the p10 gene of AcMNPV. The sequences downstream from the p10 gene in both viruses also contain long open reading frames which share homology. Northern blot analysis of RNA from OpMNPV infected O. leucostigma cells was used to define the temporal and spatial organization of transcripts from this region. S1 analysis of both termini of the major p10 mRNA indicates nontranslated regions of 52–53 bases at the 5′ end and 175 bases at the 3′ end. The 5′-mRNA start site was located within a 12-nucleotide sequence which is conserved in all late hyperexpressed baculovirus genes.