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). * The taxonomy and classification of living organisms is itself a living and steadily evolving process. The same holds true for taxonomy and classification of viruses.

The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of >/=150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica MNPV (AcMNPV) genome. More than 9% of the LdMNPV genome was occupied by 16 repeated genes related to AcMNPV ORF2. Readily identifiable homologs of several genes that have been reported to play important roles in the AcMNPV life cycle are not present; these include ie-2, a transcriptional transactivator, and gp64, a major envelope glycoprotein of the nonoccluded form of the virus. A number of genes lacking in AcMNPV but present in other baculoviruses were identified; these include two viral enhancing factor homologs, a second copy of a conotoxin-like gene, and a dutpase homolog. Although a single gene predicted to encode a large subunit of ribonucleotide reductase was found, two different copies of the small subunit gene were present. In addition, homologs of genes not previously reported for baculoviruses were identified, including a predicted protein with homology to DNA ligases and another that has motifs most closely related to a yeast mitochondrial helicase. Thirteen homologous regions (hrs) containing 54 repeated sequences that include 30-bp imperfect palindromes were identified. The imperfect palindromes are related to those from other baculoviruses.

By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa calafornica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPVpcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1.The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) is the type species of the Baculoviridae, a large family of insect viruses, and has a circular, supercoiled DNA genome of -134 kb (1, 2). It has been extensively exploited for the overexpression of eukaryotic genes and is being engineered for possible use as a viral insecticide. Despite its widespread use, little is known about the mechanism by which AcMNPV DNA replicates. Eight regions distributed around the genome have been identified as putative origins of DNA replication (3-6). Seven of these origins (ori) are located within homologous regions (hr) (Fig. 1), which contain repeats of closely related imperfect palindromes (8). One origin is located within the HindIII-K fragment, which does not contain a hr (9).To date only a putative helicase gene (10) and a putative DNA polymerase gene (11,12) have been identified as essential for baculovirus DNA replication. In addition, a gene (pcna) encoding a protein resembling proliferating-cell nuclear antigen (PCNA), which is a DNA polymerase processivity factor in other systems, has been identified in Ac-MNPV (13), but its role in DNA replication has not been determined.With a transient replication assay, six large regions of the AcMNPV genome were identified that contain one or more genes involved in DNA replication (7). In this report, this assay was used for the identification of six genes encoding proteins essential for AcMNPV DNA replication and three genes whose products stimulate DNA replication.MATERIALS AND METHODS Cells and Virus. Spodoptera frugiperda Sf9 cells (14) were cultured in TNM-FH medium (15), supplemented with 10% fetal bovine serum (FBS). The E2 strain ofAcMNPV (16) was used as wild-type virus. Routine cell culture maintenance and virus infection procedures were carried out as described (17).Plasmid Constructs. The nine replication genes were identified within six regions previously shown to be essential for DNA replication (ref. 7; see also Fig. 1). Subclones of each region were tested for their ability to substitute for the larger parental clone. The following clones were constructed. lef-) is located on the EcoRI-O fragment (18) and was cloned as an Nru I-EcoRI fragment (m.u. 7.5-8.7, ref. 2) into plasmid pUC19. lef-2 is on EcoRI-I (19, 20) and was cloned as an Mlu I fragment (m.u. 1.9-2.6) with Mlu I-Bgl II linkers into the BamHI site of pUC19. The DNA polym...

No abstract

With the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. Since less than 30% of the AcMNPV genome has been sequenced and characterized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our understanding of the baculovirus genome and its replication. The size and diversity of baculovirus genomes appears to be strongly influenced by mobile DNA from the insect host. Also, transposon-mediated mutations of baculoviruses provide examples of functional inactivation of viral genes (FP phenotype mutations) and transcriptional activation (TE-D insertion). Another role transposable elements may play is the introduction of insect promoters and enhancers to the baculovirus genome. Since early baculovirus genes are likely transcribed in a way similar to normal insect genes, transposons that insert strong constitutive promoters or cellular enhancers near early baculovirus genes may cause mutations that are subsequently selected for. If this does occur, baculovirus early gene promoters may exhibit a great deal of variability in sequence and may resemble host promoters. Given the overall similarity between the genomes of OpMNPV and AcMNPV and the apparent absence of a region, similar to the AcMNPV HindIII-K/EcoR1-S in OpMNPV, it is intriguing to speculate that this region which contains two ORFs and the hr5 enhancer, may have been inserted into the AcMNPV genome by transposition, possibly delivering several helpful genes (35k and 94k) and a powerful enhancer. The highly repeated enhancer may have been subsequently amplified by recombination. In such a model, the acquisition of general or species-specific enhancers might influence both virulence and host range. Acquisition of general enhancers could increase the level of early gene expression, thus accelerating the cellular infection cycle and making the virus more virulent. Similarly, the acquisition of species-specific enhancers might affect host range by accelerating the infection cycle, but only in a specific host or cell type. One might therefore postulate that diversity in baculoviruses may reflect not only different selection pressures but also the diversity of mobile DNA within host insect species. Although our understanding of baculovirus diversity and molecular biology is rapidly advancing, many of the fundamental characteristics that define the unique nature of baculoviruses remain poorly understood. One fundamental feature of baculoviruses is the production of the two virion phenotypes, PDV and BV.(ABSTRACT TRUNCATED AT 400 WORDS)