Effect of Temporal Expression of Integral Membrane Proteins by Baculovirus Expression Vector System.

Integral membrane proteins (IMPs) are popular target for drugs, but their resolved structures have been overlooked when compared with cytosolic proteins. The main reason is that IMPs usually need intensive post-translational modifications and they are bound to membranes, which increase the complexity of purifying or crystalizing them. Although different expression systems are used to express IMPs, baculovirus is considered one of the most successful expression systems for those proteins. Despite that, there are always unknown discrepancies in the level of IMPs expression in the baculovirus expression system. Retrospective studies have shown that expression of an immunoglobulin (anti-Chymase mouse monoclonal IgG1) driven by vp39 promoter was more efficient compared to its expression under polyhedrin (polh) promoter; however, this conclusion was not tested on different IMPs to generalize such a conclusion. In this study, the expression of eight different IMPs has been compared under vp39 and polh promoters of Autographa californica nucleopolyhedrovirus. Although different IMPs have shown different patterns of expression, the expression driven by vp39 promoter was found to be generally more efficient than the polh promoter.

On the classification and nomenclature of baculoviruses: a proposal for revision.

Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).

Identification and expression of two baculovirus gp37 genes.

The gp37 genes of the Mamestra brassicae and Lymantria dispar multicapsid nucleopolyhedroviruses (MbMNPV and LdMNPV) have been identified and characterized. Both genes were similar to other baculovirus gp37 genes and to entomopoxvirus fusolin genes. Phylogenetic analysis showed that baculovirus gp37 genes and entomopoxvirus fusolin genes form two distinct and well-separated clades. There was no evidence of recent gene transfer between the two groups. The gp37 genes also showed a distant similarity to bacterial cellulose- and chitin-binding protein genes, but the significance of this is unclear. MbMNPV and LdMNPV gp37 were both transcribed from consensus baculovirus late transcription start sites. MbMNPV gp37 was additionally transcribed from a putative early transcription start site. Tunicamycin treatment of MbMNPV-infected cells confirmed that MbMNPV GP37 is N-glycosylated. Confocal immunofluorescence microscopy revealed that the protein is located exclusively in the cytoplasm, probably in the endoplasmic reticulum.

Global protein synthesis shutdown in Autographa californica nucleopolyhedrovirus-infected Ld652Y cells is rescued by tRNA from uninfected cells.

Global protein synthesis arrest occurs in Autographa californica nucleopolyhedrovirus (AcNPV)-infected Ld652Y cells at late times postinfection (p.i.). A Lymantria dispar nucleopolyhedrovirus gene, hrf-1, precludes this protein synthesis arrest. We used in vitro translation assays to characterize the translation defect. Cell-free lysates prepared from uninfected Ld652Y cells, AcNPV-infected cells harvested at early times p.i., and cells infected with vAchrf-1, a recombinant AcNPV bearing hrf-1, all supported translation. Lysates prepared from AcNPV-infected Ld652Y cells at late times p.i. did not support translation, but activity was restored by adding small RNA species from mock-, vAchrf-1- (24 or 48 h p.i.), and AcNPV- (6 h p.i. ) infected cells. Small RNA species (24 and 48 h p.i.) from AcNPV-infected cells did not rescue translation. Assays of RNA species further fractionated by ion exchange chromatography demonstrated that tRNA rescued translation. Although specific defective tRNA species were not revealed by comparative two-dimensional gel analysis, analysis of (32)P-labeled tRNAs showed a reduction in de novo synthesis of small RNA isolated from AcNPV-infected cells compared with mock- and vAchrf-1-infected cells. This study suggests a mechanism of translation arrest involving defective or depleted tRNA species in AcNPV-infected Ld652Y cells.

Lymantria dispar nucleopolyhedrovirus hrf-1 expands the larval host range of Autographa californica nucleopolyhedrovirus.

The gypsy moth (Lymantria dispar) is nonpermissive for Autographa californica nucleopolyhedrovirus (AcNPV) infection. We previously isolated a gene, host range factor 1 (hrf-1), from L. dispar nucleopolyhedrovirus that promotes AcNPV replication in Ld652Y cells, a nonpermissive L. dispar cell line (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996). In the present study, we investigated the ability of hrf-1 to alter the larval host range of AcNPV. Bioassays using recombinant AcNPV bearing hrf-1 were conducted with insect larvae by use of oral infection. AcNPV bearing hrf-1 was infectious for neonate L. dispar larvae, with a 50% lethal concentration of 1.2 x 10(5) polyhedral inclusion bodies/ml of diet, which is similar to that of wild-type AcNPV for permissive hosts. AcNPV can kill neonate L. dispar larvae at high doses, but it does not kill third-instar larvae. However, electron microscopy studies of AcNPV-inoculated third-instar larvae revealed virus replication in the midgut cells. PCR analyses indicated that the virus was AcNPV. These results suggest that the block for AcNPV infection of L. dispar larvae is its inability to spread systematically from primary infection sites in the midgut epithelium and that this barrier is leaky in neonates. hrf-1 allows AcNPV to overcome this barrier. AcNPV recombinants bearing hrf-1 were also significantly more infectious for Helicoverpa zea, a resistant species, suggesting that the blocks for AcNPV infection of L. dispar and H. zea larvae may be similar.

Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection.

Prospects for altering host range for baculovirus bioinsecticides.

Advances in the understanding of baculovirus replication and the identification of genes that affect host range set the stage for constructing recombinant baculoviruses for specific past insects. The modification of baculovirus host specificity has recently been achieved by inserting or deleting genes that affect virus replication or cellular defenses.

Characterization of host range factor 1 (hrf-1) expression in Lymantria dispar M nucleopolyhedrovirus- and recombinant Autographa californica M nucleopolyhedrovirus-infected IPLB-Ld652Y cells.

We previously identified a gene, host range factor 1 (hrf-1), in Lymantria dispar M nucleopolyhedrovirus (LdMNPV) which promoted Autographa californica M nucleopolyhedrovirus (AcMNPV) replication in a nonpermissive cell line IPLB-Ld652Y (Ld652Y). A recombinant AcMNPV, vAcLdPS, that bore hrf-1 controlled by two synthetic baculovirus late promoters and that replicated in Ld652Y cells was constructed. In this study, we constructed a new recombinant AcMNPV, vAcLdPD, bearing only hrf-1 controlled by its own promoter. vAcLdPD replicated in Ld652Y cells in the same manner as vAcLdPS, confirming that hrf-1 alone was sufficient to promote AcMNPV replication in Ld652Y cells. hrf-1 was transcribed as a delayed early gene in LdMNPV but as an immediate early gene in both recombinant AcMNPVs. Primer extension analysis showed that the initiator sequence TCAGT was used as the transcription start site in both LdMNPV and recombinant AcMNPVs. Additional sequencing revealed several regulatory motifs in the hrf-1 upstream region. hrf-1 transcripts in LdMNPV- and vAcLdPS-infected Ld652Y cells terminated near the polyadenylation signal at the end of hrf-1 ORF while in vAcLdPD, the hrf-1 transcripts terminated at a downstream polyadenylation signal at the end of ORF 603. Using Western blot analysis, we detected HRF-1 expression in both recombinant AcMNPV-infected Ld652Y cells but not in LdMNPV-infected Ld652Y cells.

Differential infectivity of two Autographa californica nucleopolyhedrovirus mutants on three permissive cell lines is the result of lef-7 deletion.

We isolated two Autographa californica nucleopolyhedrovirus (AcMNPV) mutants that have infectivity similar to that of wild-type (wt) AcMNPV in TN368 cells, but reduced budded virus and polyhedral inclusion body production in IPLB-SF-21 and SE1c cells. Restriction endonuclease analysis and sequence analysis indicated that 3.2-kb (77.0-79.4 m.u.) and 4.4-kb (76.7-80.1 m.u.) regions, the location of four major open reading frames (ORFs), pk2, ORF-247, lef-7, and chitinase, were deleted in mutant T295 and T297, respectively. Phenotypes of recombinant viruses vdel-AG, in which all four ORFs were deleted, and vlef7-AG, in which only lef-7 was deleted, were identical to the mutants. The phenotypes of recombinant viruses with deletions of the other ORFs were indistinguishable from wt AcMNPV. This demonstrated that the deletion of lef-7 was responsible for the mutant phenotypes. Viral DNA synthesis in both mutant- and vlef7-AG-infected SF-21 and SE1c cells was reduced to less than 10% of that of wt AcMNPV-infected cells. In TN368 cells, DNA synthesis in mutant- and vlef7-AG-infected cells was delayed relative to wt-infected cells. Although lef-7 is not essential for AcMNPV infection in TN368 cells, it is expressed in TN368, SF-21, and SE1c cells in a similar manner.

Identification of baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.

A gene that promotes Autographa californica M nuclear polyhedrosis virus (AcMNPV) replication in IPLB-Ld652Y cells, a cell line that is nonpermissive for AcMNPV, was identified in Lymantria dispar M nuclear polyhedrosis virus (LdMNPV). Cotransfection of AcMNPV DNA and a plasmid carrying the LdMNPV gene into IPLB-Ld652Y cells results in AcMNPV replication. The gene maps between 43.3 and 43.8 map units on the 162-kbp genome of LdMNPV. It comprises a 218-codon open reading frame and encodes a polypeptide with a predicted molecular mass of 25.7 kDa. The predicted polypeptide is glutamic acid and valine rich and negatively charged, with a pI of 4.61. No protein sequence motifs were identified, and no matches with known nucleotide or peptide sequences were found in the AcMNPV genome or database searches that suggest how this gene might function. A recombinant AcMNPV bearing the LdMNPV gene overcomes a block in protein synthesis observed in AcMNPV-infected IPLB-Ld652Y cells. Using Southern blotting techniques, we were unable to identify a homolog in Orgyia pseudotsugata M nuclear polyhedrosis virus, a baculovirus that is routinely propagated in IPLB-Ld652Y cells. This suggests that the LdMNPV host range is unique among the baculoviruses studied to date. We named this gene hrf-1 (for host range factor 1).

Physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus.

A physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus (AfMNPV) DNA was constructed for restriction endonucleases EcoRI, HindIII, PstI and XhoI. The genome size was estimated to be 130 kbp. Ordering of the restriction fragments was accomplished by cross-blot hybridization, double digestion and DNA-DNA hybridization. The polyhedrin gene and homologous repeat (hr) regions were located by hybridization to the Autographa californica MNPV (AcMNPV) polyhedrin gene and hr4, respectively. Restriction pattern comparison and Southern blot analysis suggest that AfMNPV is closely related to AcMNPV.

Tracking microbial populations effective in reducing exposure.

Microbial ecology provides the link between basic biochemical and molecular studies on toxicity reduction by microbial metabolism and environmental studies that determine exposure. This link provides the ability to determine which microorganisms are responsible for the actual transformations in nature, thereby establishing how predictive the laboratory pathway, kinetic, regulatory, and enzyme mechanistic information is for nature. This information can be important to the rate of toxicant removal, the type and concentration of intermediate product(s), and the identification of conditions that limit effective toxicant removal. Nucleic acid-based methods now provide the main means to track important biodegrading populations. Examples of these methods are given that illustrate tracking a biodegrading microbe injected into an aquifer, following community succession in a toluene-degrading fluidized bed reactor, aiding the isolation from nature of novel biodegrading organisms, and rapidly characterizing the extent of microbial diversity in an aquifer stimulated to co-metabolize trichloroethene.

Use of molecular techniques to evaluate the survival of a microorganism injected into an aquifer.

A PCR primer set and an internal probe that are specific for Pseudomonas sp. strain B13, a 3-chlorobenzoate-metabolizing strain, were developed. Using this primer set and probe, we were able to detect Pseudomonas sp. strain B13 DNA sequences in DNA extracted from aquifer samples 14.5 months after Pseudomonas sp. strain B13 had been injected into a sand and gravel aquifer. This primer set and probe were also used to analyze isolates from 3-chlorobenzoate enrichments of the aquifer samples by Southern blot analysis. Hybridization of Southern blots with the Pseudomonas sp. strain B13-specific probe and a catabolic probe in conjunction with restriction fragment length polymorphism (RFLP) analysis of ribosome genes was used to determine that viable Pseudomonas sp. strain B13 persisted in this environment. We isolated a new 3-chlorobenzoate-degrading strain from one of these enrichment cultures. The B13-specific probe does not hybridize to DNA from this isolate. The new strain could be the result of gene exchange between Pseudomonas sp. strain B13 and an indigenous bacterium. This speculation is based on an RFLP pattern of ribosome genes that differs from that of Pseudomonas sp. strain B13, the fact that identically sized restriction fragments hybridized to the catabolic gene probe, and the absence of any enrichable 3-chlorobenzoate-degrading strains in the aquifer prior to inoculation.

Differential gene expression mediated by late, very late and hybrid baculovirus promoters.

A series of recombinant viruses was constructed to study the regulation of expression from the promoter (Pcap) for the major capsid-protein-encoding gene (vp39) of the baculovirus, Autographa californica nuclear polyhedrosis virus. Each virus of this series contains the cat reporter gene under the control of Pcap or portions thereof. Pcap regulation of cat was determined at the RNA and enzyme activity levels, and was compared to cat gene expression driven by Ppolh, the promoter of the polyhedrin gene (polh). Although optimum expression was achieved with a 464-bp region, which included all three Pcap transcription start points (tsp), a 99-bp segment of Pcap containing a single tsp was sufficient to direct late cat expression. Ppolh and Pcap-mediated regulation differed temporally; Pcap-cat constructs were expressed at 12 h post-infection (p.i.) and continued through 48 h p.i., whereas Ppolh-cat were not expressed at 12 h, but were initiated around 18 h p.i. and underwent a burst of expression between 24 and 48 h p.i. A recombinant virus carrying a hybrid and polh promoter (Pcappolh) was also constructed and studied; Pcappolh contained the two distal tsp of Pcap and a proximal tsp of Ppolh. Pcappolh exhibited both 'late' and 'very late' regulation; the distal tsp were regulated as late sites and the proximal tsp was regulated primarily as a very late site although it also showed a weak late response. Hybrid late/very late promoters should prove useful in optimizing foreign gene expression using baculovirus vectors.

A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper.

An Autographa californica nuclear polyhedrosis virus gene encoding a 30-kilodalton polypeptide with two different sequence motifs characteristic of DNA-binding proteins was identified immediately downstream of the major capsid protein gene (vp39). The gene, CG30, was characterized by sequencing, transcriptional mapping, in vitro translation of hybrid-selected RNA, and comparison of the derived polypeptide sequence with published data bases. The initial ATG of the 792-base-pair CG30 open reading frame is two nucleotides downstream of the vp39 terminal TAA codon. Early transcripts of CG30 initiate within the vp39 coding sequence. At late times, bicistronic transcripts initiate from the vp39 promoter, continue through CG30, and terminate at the same site as the early transcripts. In vitro translation of hybrid-selected early CG30 RNA yields a polypeptide of 30 kilodaltons. The predicted CG30 polypeptide sequence has characteristics of a eucaryotic transcriptional activator and is novel in having two potential DNA-binding domains. A stretch of acidic residues bridges a zinc finger at the amino terminus and a leucine zipper with a flanking basic region at the carboxyl terminus.

Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus.

The gene encoding the major capsid protein of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) was identified, sequenced, and transcriptionally mapped. The location of the gene was determined by immunological screening of an expression library of AcMNPV open reading frame-beta-galactosidase fusions with an antibody raised to virus structural proteins. The DNA sequence of the corresponding region, which mapped within 56.6 and 58.0 map units on the AcMNPV genome, revealed a 1,040-base-pair open reading frame capable of encoding a 39-kilodalton polypeptide. The identity of the polypeptide was determined by Western blot (immunoblot) analysis of purified empty capsids with an antibody raised to the capsid-beta-galactosidase fusion protein. The identity of the peptide encoded by the gene was confirmed by immunoprecipitation of an in vitro translation product with RNA selected by hybridization to DNA sequences from the coding region of the gene. Transcripts of the capsid gene were analyzed by Northern (RNA) blots and mapped by nuclease protection and primer extension analysis. The capsid gene is transcribed maximally at 12 and 24 h postinfection but not in the presence of cycloheximide, a protein synthesis inhibitor, or aphidicolin, a viral DNA synthesis inhibitor, and is therefore classified as a late gene. The gene is transcribed in a counterclockwise direction with respect to the circular map. There are three transcriptional start sites, all containing the AGTAAG consensus sequence found at the start site of all late AcMNPV genes.