<Emphasis Type="Italic">vlf-1</Emphasis> Deletion Brought Ac<Emphasis Type="Italic">M</Emphasis>NPV to Defect in Nucleocapsid Formation

Recent studies have provided direct evidence that the baculovirus very late factor 1 (VLF-I) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was essential for BV production. To elucidate how vlf-1 deletion blocks BV production we generated a vlf-1 knockout bacmid by ET-recombination technology on AcMNPV bacmid propagated in Escherichia coli. Bacmid DNA transfection and supernatant passage assay revealed that the vlf-1 knockout bacmid was unable to replicate in cell culture, while vlf-1 repair bacmid, which was generated by transposition of the vlf-1 ORF under control of its native promoter into polyhedrin gene locus of vlf-1 knockout bacmid, resumed viral replication ability at wildtype levels. Results of these assays proved the correct construction of the vlf-1 knockout bacmid. Subsequent electron microscopy revealed that the vlf-1 knockout bacmid failed to form nueleocapsid in the nuclei of the transfected cells. Instead, intensely electron-dense virogenic stroma characteristic of viral DNA synthesis were observed. Thus, it is demonstrated for the first time that vlf-1 knockout blocked nucleocapsid formation and the defective nucleocapsid formation resulted in the abolishment of BV and ODV production. Possible roles of vlf-1 in genome processing are suggested and discussed.     

A highly conserved baculovirus gene p48 (ac103) is essential for BV production and ODV envelopment

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc^{P48-KO-PH-GFP} was unable to propagate in cell culture, while a ‘repair’ Bacmid vAc^{P48-REP-PH-GFP} was able to replicate in a manner similar to a wild-type Bacmid vAc^PH-GFP. Titration assays and Western blotting confirmed that vAc^{P48-KO-PH-GFP} was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.     

Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc^{ac53KO-PH-GFP}) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc^{ac53KO-PH-GFP} could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are present in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production.     

Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus, general synthesis of preoccluded virions and occlusion body formation

Although orf66 (ac66) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in all sequenced lepidopteran baculovirus genomes, its function is not known. This paper describes generation of an ac66 knockout AcMNPV bacmid mutant and analyses of the influence of ac66 deletion on the virus replication in Sf-9 cells so as to determine the role of ac66 in the viral life cycle. Results indicated that budded virus (BV) yields were reduced over 99% in ac66-null mutant infected cells in comparison to that in wild-type virus infected cells. Optical microscopy revealed that occlusion body synthesis was significantly reduced in the ac66 knockout bacmid-transfected cells. In addition, ac66 deletion interrupted preoccluded virion synthesis. The mutant phenotype was rescued by an ac66 repair bacmid. On the other hand, real-time PCR analysis indicated that ac66 deletion did not affect the levels of viral DNA replication. Electron microscopy revealed that ac66 is not essential for nucleocapsid assembly, but for the efficient transport of nucleocapsids from the nucleus to the cytoplasm. These results suggested that ac66 plays an important role for the efficient exit of nucleocapsids from the nucleus to the cytoplasm for BV synthesis as well as for preoccluded virion and occlusion synthesis.     

A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.     

Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame

To investigate the role of the gene products encoded from the open reading frames 101, 142, and 144 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a set of bacmid knockout and repair constructs were generated. The repair genes were engineered to contain an HA epitope tag at their C-termini. The results of transfection-infection assays and growth curve analyses showed that the Ac 101, 142, and 144 genes were required for infectious virus production. To better characterize the role of these genes in the baculovirus replication cycle, quantitative DNA replication assays were performed and demonstrated that in cells transfected with the Ac 101, 142, or 144 knockouts, DNA replicated with similar kinetics as a control virus. Western blot analyses of budded virus from cells infected with the repair viruses showed that these proteins are associated with the viral nucleocapsid. Furthermore, immunoelectron microscopy of cells transfected with the knockout bacmids revealed defects in nucleocapsid production for all three constructs. From these results we concluded that the gene products encoded from these open reading frames are essential for virus production and may be involved in DNA processing, packaging, or nucleocapsid morphogenesis.     

Influence of codon usage and translation initiation codon context in theAcNPV-based expression system: Computer analysis using homologous and heterologous genes

Codon usage by all the known gene sequences fromAutographa californica nuclear polyhedrosis virus (AcNPV) was compared with that of firefly luciferase (luc) and the beta subunit of human chorionic gonadotropin (hCG) expressed to contrasting levels in the baculovirus system. The highly expressedluc gene showed a codon usage similar toAcNPV genes, as reflected by a very low D-squared statistic value (0.78) and a similar G/C usage (45%) at wobble positions. However, the underexpressed hCG gene displayed a high D-squared value (7.3) and G/C usage (82.5%) at the wobble base positions. Alignment of the 20 nucleotides around the initiation codon of 23AcNPV genes identified a novel consensus translation initiation sequence aag/ta/tat/aa/cAAaATGaa/ct/ag/aAan, which was quite different from the Kozak consensus sequence (GCC)GCCA/GCCATGG. An extension of these analyses to a sample of other heterologous genes overexpressed and underexpressed in BEVS suggested similar trends. These theoretical analyses have important implications for heterologous gene expression in this system.     

Identification and nucleotide sequence of an ecdysteroid UDP-glucosyltransferase gene ofSpodoptera littoralis multicapsid nuclear polyhedrosis virus

TheSpodoptera littoralis multicapsid nuclear polyhedrosis virus (SlMNPV) is a member of the Baculoviridae that shows a distant genetic relationship to the prototypeAutographa californica MNPV (AcMNPV). Using anAcMNPV gene-specific probe, we identified and mapped an ecdysteroid UDP-glucosyltransferase (egt) gene in the genome ofSlMNPV. Sequence determination of a part from the hybridizing DNA fragment revealed an open reading frame of 1548 nucleotides that exhibits 38% and 44% identity to theegt amino acid sequences ofAcMNPV andLymantria dispar MNPV (LdMNPV), respectively. Sequences flanking theSlMNPVegt gene, including the promoter region, were found to be unique to the virus. The presence of this nonstructural gene inSlMNPV and several other baculoviruses points to the importance ofegt for the viral infection process.     

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines

Summary.  We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef-6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV. Received May 3, 2002; accepted September 19, 2002     

<Emphasis Type="Italic">Autographa californica</Emphasis> multiple nucleopolyhedrovirus <Emphasis Type="Italic">odv-e25</Emphasis> (Ac94) is required for budded virus infectivity and occlusion-derived virus formation

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e25 is a core gene found in all lepidopteran baculoviruses, but its function is unknown. In this study, we generated an odv-e25-knockout AcMNPV and investigated the roles of ODV-E25 in the baculovirus life cycle. The odv-e25 knockout was subsequently rescued by reinserting the odv-e25 gene into the same virus genome. Fluorescence microscopy showed that transfection with the odv-e25-null bacmid vAcBac^KO was insufficient for propagation in cell culture, whereas the ‘repair’ virus vAcBac^RE was able to function in a manner similar to that of the control vAcBac. We found that odv-e25 was not essential for the release of budded viruses (BVs) into culture medium, although the absence of odv-e25 resulted in a 100-fold lower viral titer at 24 h post-transfection (p.t.). Analysis of viral DNA replication in the absence of odv-e25 showed that viral DNA replication was unaffected in the first 24 h p.t. Furthermore, electron microscopy revealed that polyhedra were found in the nucleus, while mature occlusion-derived viruses (ODVs) were not found in the nucleus or polyhedra in odv-e25 null transfected cells, which indicated that ODV-E25 was required for the formation of ODV.     

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10⁹ infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.     

Molecular Anatomy of Chilo Iridescent Virus Genome and the Evolution of Viral Genes

Chilo iridescent virus (CIV) or Insect iridescent virus 6 (IIV-6) is the type species of the genus iridovirus, a member of the Iridoviridae family. CIV is highly pathogenic for a variety of insect larvae and this implicates a possible use as a biological insecticide. CIV progeny and assembly occur in the cytoplasm of the infected cell and accumulate in the fatbody of the infected insects. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure and the amino acid sequences of different viral gene products. The elucidation of the coding capacity and strategy of CIV was the first step towards understanding the underlying mechanisms of viral infection, replication and virus-host interaction. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity of the CIV genome was determined by the analysis of the complete DNA nucleotide sequence consisting of 212,482bp that represent 468 open reading frames encoding for polypeptides ranging from 40 to 2432 amino acid residues. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were non-overlapping. The identification of several putative viral gene products including a DNA ligase and a viral antibiotic peptide is a powerful tool for the investigation of the phylogenetic relatedness of this evolutionary and ecologically relevant eukaryotic virus.     

Identification and Classification of the Spodoptera littoralis Nucleopolyhedrovirus Inhibitor of Apoptosis Gene

Baculoviruses possess two types of genes that suppressed apoptosis, p35 and inhibitor of apoptosis (iap). In this study we report the isolation and identification of an inhibitor of apoptosis gene Sliap in the genome of the Spodoptera littoralis nucleopolyhedrovirus (SlNPV). The Sliap sequence predicted a 15 kDa polypeptide with only one BIR domain and a RING finger, both motifs characteristic of the IAP family of proteins, and a third specific acidic-rich motif. These characteristics, shared with the Spodoptera littura NPV IAP2/3, Epiphyas postvittana NPV IAP4, Lymantria dispar NPV IAP and Orgyia pseudotsugata NPV IAP4 (Orf 107) allowed us to classify them in a new homology group (IAP-4). Sliap was able to delay, but not to suppress, apoptosis induced by replication of a recombinant AcMNPV deficient in p35. In SlNPV infected-SF9 cells Sliap was expressed earlier than sl-p49 suggesting that its role at the initiation of infection was to delay the apoptotic response of the host.     

Genetic analysis of a host determination mechanism of bromoviruses in Arabidopsis thaliana

Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins.