George F. Rohrmann

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).

Baculovirus structural proteins

Introduction. Baculovirus biology. The Baculoviridae are a diverse family of virus pathogens which are infectious for arthropods, particularly insects of the order Lepidoptera. They have also been isolated from the insect orders Hymenoptera, Diptera and Trichoptera, as well as from the crustacean order Decapoda (shrimp) (Couch, 1974). Although baculoviruses infect over 600 species of insects (Martignoni & Iwai, 1986), individual isolates normally show a limited host range and infect only closely related species. Baculoviruses are characterized by large rod-shaped virions containing supercoiled dsDNA genomes ranging in size from 88 to over 160 kbp. A prominent feature is the occlusion of virions in a crystalline protein matrix, and two genera have been distinguished based on the gross structure of the occlusion body. One genus, the nuclear polyhedrosis viruses (NPVs), has polyhedron-shaped occlusion bodies 1 to 15 µm in diameter composed of a protein called polyhedrin which crystallizes around many enveloped nucleocapsids (see Fig. 1 and 2).

Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae.

ABSTRACT The complete genome sequences of a number of diverse members of theBaculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected withld130-egfp. In addition, cells transfected with eitherld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution ofgp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.

REPLICATION OF BACULOVIRUS DNA

Introduction. The Baculoviridae is a diverse family of pathogens that are infectious for arthropods and are characterized by a complex replication cycle that culminates in the occlusion of virions in a crystalline protein matrix (Blissard & Rohrmann, 1990). Over 400 lepidopteran species serve as hosts for these viruses, with a single virus isolate usually restricted to one or a few related species. In addition, they have been reported from species of the orders Hymenoptera, Diptera, Siphonoptera and Trichoptera, as well as in several crustaceans (Adams & McClintock, 1991; Martignoni & Iwai, 1986). Baculoviruses are divided into two genera based on occlusion body morphology; the nuclear polyhedrosis viruses (NPVs) (Rohrmann, 1994), which are characterized by many virions present in each polyhedron-shaped occlusion body, and the granulosis viruses (GVs) (Crook, 1994), which normally have smaller occlusion bodies each containing only a single virion, and have been reported to infect only the Lepidoptera.

Characterization of the Role of Very Late Expression Factor 1 in Baculovirus Capsid Structure and DNA Processing

ABSTRACT Very late expression factor 1 (VLF-1) of Autographa californica multiple nucleopolyhedrovirus is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production. In this report, we show that a vlf-1 knockout bacmid was able to synthesize viral DNA at levels similar to that detected for a gp64 knockout bacmid that served as a noninfectious control virus. Additionally, analysis of replicated bacmid DNA by field-inversion gel electrophoresis indicated that VLF-1 is not required for synthesizing high-molecular-weight intermediates that could be resolved into unit-length genomes when cut at a unique restriction site. However, immunoelectron microscopic analysis revealed that in cells transfected with a vlf-1 knockout bacmid, aberrant tubular structures containing the capsid protein vp39 were observed, suggesting that this virus construct was defective in producing mature capsids. In contrast, rescuing the vlf-1 knockout bacmid construct with a copy of VLF-1 that carries a mutation of a highly conserved tyrosine (Y355F) was sufficient to restore the production of nucleocapsids with a normal appearance, but not infectious virus production. Furthermore, the results of a DNase I protection assay indicated that the DNA packaging efficiency of the VLF-1(Y355F) virus construct was similar to that of the gp64 knockout control. Finally, a recombinant virus containing a functional hemagglutinin epitope-tagged version of VLF-1 was constructed to investigate the association of VLF-1 with the nucleocapsid. Analysis by immunoelectron microscopy of Sf-9 cells infected with this virus showed that VLF-1 localized to an end region of the nucleocapsid. Collectively, these results indicate that VLF-1 is required for normal capsid assembly and serves an essential function during the final stages of the DNA packaging process.

Transfer, Incorporation, and Substitution of Envelope Fusion Proteins among Members of the Baculoviridae, Orthomyxoviridae, and Metaviridae (Insect Retrovirus) Families†

Recent research suggests that members of the Baculoviridae family can be divided into two groups on the basis of their envelope fusion proteins (31). One group utilizes proteins related to GP64. Homologs of GP64 are also used by the thogotoviruses (27), a genus of the Orthomyxoviridae family. Members of the other group of baculoviruses utilize envelope fusion proteins related to a protein called LD130. LD130 has been shown to be related to the envelope protein of insect retroviruses in the genus Errantivirus (family Metaviridae) (24, 36). In this review, the evidence for these data is outlined and possible pathways of transfer, incorporation, and substitution are discussed.

Baculovirus DNA Replication

Baculovirus replication initiates a cascade of gene expression that ultimately results in the production of progeny virus. This cascade is regulated at different points during the replication cycle: early gene expression is regulated by the interaction of cis-acting elements, viral trans-acting factors, and host factors (see Chapter 6, this volume), and late/very late gene expression is regulated by a combination of viral DNA replication, cis-acting elements as well as early and late viral factors (see Chapter 8, this volume).

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 promoter 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.

Relatedness of baculovirus and gypsy retrotransposon envelope proteins

BackgroundCurrent evidence suggests that lepidopteran baculoviruses may be divided into two phylogenetic groups based on their envelope fusion proteins. One group utilizes gp64, a low pH-dependent envelope fusion protein, whereas the other employs a protein family (e.g. LD130 in the Lymantria dispar nucleopolyhedrovirus) unrelated to gp64, but that is also low pH-dependent. Database searches with members of the LD130 protein family often record significant levels of homology to envelope proteins from a number of insect retrovirus-like transposable elements of the gypsy class. In this report, the significance of the homology between these two types of envelope proteins is analyzed.ResultsThe significance of the alignment scores was evaluated using Z-scores that were calculated by comparing the observed alignment score to the distribution of scores obtained for alignments after one of the sequences was subjected to 100 random shuffles of its sequence. These analyses resulted in Z-scores of >9 for members of the LD130 family when compared to most gypsy envelope proteins. Furthermore, in addition to significant levels of sequence homology and the presence of predicted signal sequences and transmembrane domains, members of this family contain a possible a furin cleavage motif, a conserved motif downstream of this site, predicted coiled-coil domains, and a pattern of conserved cysteine residues.ConclusionsThese analyses provide a link between envelope proteins from a group of insect retrovirus-like elements and a baculovirus protein family that includes low-pH-dependent envelope fusion proteins. The ability of gypsy retroelements to transpose from insect into baculovirus genomes suggests a pathway for the exchange of this protein between these viral families.

Localization of the 34-kDa polyhedral envelope protein in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus.

SummaryUsing immuno-electron microscopy the 34 kDa polyhedron envelope (PE) phosphoprotein (pp 34) was localized in cells infected withAutographa californica multiple-nucleocapsid nuclear polyhedrosis virus (AcMNPV). In wild-type AcMNPV-infected cells this protein was found associated with electron-dense “spacers” and the polyhedron envelopes demonstrating their structural relationship. In these cells pp 34 was also found associated with fibrillar structures present in the nucleus and cytoplasm of infected cells. However, when cells were infected with an AcMNPV mutant with an inactivated pp 34 gene, antiserum against pp 34 still localized in fibrillar structures. In cells infected with AcMNPV mutants lacking p 10, and thus devoid of fibrillar structures, pp 34 localized normally with both electron-dense “spacers” and polyhedron envelopes. These data confirm that fibrillar structures are not essential for the morphogenesis of polyhedron envelopes and demonstrate that the association of anti pp 34 serum with these structures is specific but fortuitous.