David A. Theilmann

On the classification and nomenclature of baculoviruses: A proposal for revision

Summary.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).

Molecular analysis of Campoletis sonorensis virus DNA in the lepidopteran host Heliothis virescens

Nucleic acid hybridization techniques were used to analyse the fate of Campoletis sonorensis virus (CsV) DNAs in naturally parasitized and virus-injected Heliothis virescens larvae. Viral DNA persisted in injected H. virescens larvae from 0 to 10 days post-injection but no increase in the amount of viral DNA could be detected. Similarly, no increase in the amount of viral DNA was detected in naturally parasitized H. virescens larvae before the development of the C. sonorensis pupae. However, a dramatic increase of viral DNA was detected in pharate and newly emerged (0 to 48 h) adult C. sonorensis wasps. The results from these in vivo molecular analyses suggest that CsV replication does not occur in naturally parasitized or virus-injected H. virescens and that viral replication may be restricted to tissues of C. sonorensis wasps.

Analysis of the insect os-d-like gene family.

Insect OS-D-like proteins, also known as chemosensory (CSP) or sensory appendage proteins (SAP), are broadly expressed in various insect tissues, where they are thought to bind short to medium chain length fatty acids and their derivatives. Although their specific function remains uncertain, OS-D-like members have been isolated from sensory organs (including the sensillum lymph in some cases), and a role in olfaction similar to that of the insect odorant binding proteins (OBP) has been suggested for some. We have identified 15 new OS-D-like sequences: four from cDNA clones described herein and 11 from sequence databases. The os-d-like genes from the Anopheles gambiae, Apis mellifera, Drosophila melanogaster, and Drosophila pseudoobscura genomes typically have single, small introns with a conserved splice site. Together with all family members entered on GenBank, a total of 70 OS-D-like proteins, representing the insect orders Diptera, Dictyoptera, Hymenoptera, Lepidoptera, Orthoptera, and Phasmatodea, were analyzed. A neighbor joining distance phenogram identified several protein similarity classes that were characterized by highly conserved sequence motifs, including (A) N-terminal YTTKYDN(V/I)(N/D)(L/V)DEIL, (B) central DGKELKXX(I/L)PDAL, and (C) C-terminal KYDP. In contrast, three similarity classes were characterized by their diversion from these conserved motifs. The functional importance of conserved amino acid residues is discussed in relation to the crystal and NMR structures of MbraCSPA6.

Baculovirus immediate-early promoter-mediated expression of the Zeocin™ resistance gene for use as a dominant selectable marker in Dipteran and Lepidopteran insect cell lines

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 microg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 microg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3-4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by beta-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.

Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc96null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc96null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc96null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).

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.

Identification and comparison ofCampoletis sonorensis virus transcripts expressed from four genomic segments in the insect hostsCampoletis sonorensis andHeliothis virescens

The Campoletis sonorensis virus (CsV; Polydnaviridae) genome consists of at least 28 closed circular superhelical (SH) DNAs. In this study we used complete clones of four SH DNAs to analyze viral transcription both in the adult parasitic wasp host Campoletis sonorensis (Ichneumonidae) and in the lepidopteran host, Heliothis virescens (Noctuidae). CsV genes are expressed in parasitized H. virescens, but no viral transcripts had been characterized from C. sonorensis until this study. The clones of the SH DNAs B, H, M, and O1 were used to probe Northern blots of poly(A)+ RNA isolated from C. sonorensis reproductive tissue and from parasitized H. virescens larvae. All four SH DNAs hybridized to viral transcripts. SH-H,-M, and -O1 hybridized to messages expressed in both hosts. SH-B and -M hybridized to transcripts that were detected only in either C. sonorensis reproductive tissue or parasitized H. virescens larvae. These results suggest that some CsV genes are expressed in a host-specific manner. In a previous study we identified a family of imperfectly conserved tandemly repeated 540-bp repeat elements on SH-B,-H and -O1 (D. A. Theilmann and M. D. Summers, 1987 J. Virol. 61; 2589-2598). Hybridization of the 540-bp repeat regions to Northern blots showed that they were all homologous to viral transcripts. A cDNA clone of a mRNA that is transcribed from the 540-bp repeat region of SH-B was isolated from a lambda gt 10 library and completely sequenced. The sequence data revealed that the 540-bp repeat element was contained within the open reading frame of this gene. These results indicate that transcribed sequences homologous to the 540-bp repeat elements represent a second gene family to be identified within the CsV genome.

Autographa californica Multiple Nucleopolyhedrovirus exon0 (orf141), Which Encodes a RING Finger Protein, Is Required for Efficient Production of Budded Virus

ABSTRACT exon0 (orf141) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene that codes for a predicted 261-amino-acid protein. Located in the C-terminal region of EXON0 are a predicted leucine-rich coiled-coil domain and a RING finger motif. The 5′ 114 nucleotides of exon0 form part of ie0, which is a spliced gene expressed at very early times postinfection, but transcriptional analysis revealed that exon0 is transcribed as a late gene. To determine the role of exon0 in the baculovirus life cycle, we used AcMNPV bacmids and generated exon0 knockout viruses (Ac-exon0-KO) by recombination in Escherichia coli. Ac-exon0-KO progressed through the very late phases in Sf9 cells, as evidenced by the development of occlusion bodies in the nuclei of the transfected or infected cells. However, production of budded virus (BV) in Ac-exon0-KO-infected cells was reduced at least 3 orders of magnitude in comparison to that in wild-type virus infection. Microscopy revealed that Ac-exon0-KO was restricted primarily to the cells initially infected, exhibiting a single-cell infection phenotype. Slot blot assays and Western blot analysis indicated that exon0 deletion did not affect the onset or levels of viral DNA replication or the expression of IE1, IE0, and GP64 prior to BV release. These results demonstrate that exon0 is required for efficient production of BV in the AcMNPV life cycle but does not affect late occlusion-derived virus.

A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines

A series of shuttle vectors have been constructed that allow expression of heterologous proteins in either dipteran or lepidopteran insect cell lines. Constitutive expression in a broad range of host cells is mediated by the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) immediate-early 2 (ie2) promoter. Alternatively, if inducible expression is required, for example to express cytotoxic proteins, a vector has been constructed that uses the Drosophila metallothionein (Mtn) promoter for metal-inducible protein expression in dipteran cell lines. A chimeric synthetic bacterial-OpMNPV ie promoter-Zeocin resistance gene cassette has been included to facilitate cloning in E. coli as well as the generation of stably transformed insect cell lines. The utility of the system is demonstrated by the constitutive and inducible expression of the highly processed glycosylphosphatidylinositol-anchored glycoprotein, human melanotransferrin, in transformed insect cell lines.

Segment W of Campoletis sonorensis virus: Expression, gene products, and organization

Campoletis sonorensis virus (CsV, Polydnaviridae) is a segmented double-stranded DNA virus which has an apparently symbiotic relationship with the parasitic wasp, Campoletis sonorensis. CsV replicates in the oviducts of the parasitic wasp and is injected into the wasps host, Heliothis virescens (Lepidoptera; Noctuiidae), during oviposition. In the parasitized lepidopteran host, the virus has a dramatic effect on host physiology and viral gene products are believed to play an essential role in the survival of the parasitic wasps egg and larva. In the current study, we used Northern blot analyses to examine expression from segment W in the parasitized host and in the parasitic wasp. Segment W hybridized primarily to two relatively abundant mRNAs (1.6 and 1.0 kb) from the parasitized host. These 1.6- and 1.0-kb mRNAs, which were previously shown to be transcribed from two closely related genes (WHv1 and WHv2) on segment W (G. W. Blissard, O. P. Smith, and M. D. Summers, 1987, Virology 160, 120-134) increased in relative abundance between 2 and 24 hr postparasitization (pp) and were detected throughout parasitization (8 days). To study the proteins encoded by these closely related genes, the open reading frame from each of the related genes was cloned into a baculovirus expression vector. By pulse labeling in the presence and absence of tunicamycin, we examined secretion and glycosylation of these CsV proteins in infected lepidopteran cells (Spodoptera frugiperda). Expression of segment W in the oviducts of the female wasp was also examined. Segment W hybridized to at least five CsV mRNAs on Northern blots of poly(A) mRNA from C. sonorensis oviducts. To identify specific CsV mRNAs and map putative viral genes expressed in wasp oviduct tissues, segment W was used to screen a cDNA library of C. sonorensis oviduct mRNAs. Three cDNAs were used to identify CsV mRNAs by Northern blot analyses and to map the locations of three putative CsV genes on segment W. Cross-hybridization within the CsV genome was examined with cloned segment W and with the three cloned cDNAs.