Transcription and promoter analysis of pif, an essential but low-expressed baculovirus gene

The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-B⌬16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-B⌬pifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-B⌬16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-B⌬16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-B⌬pifs were almost identical to the results observed with SfNIC-B⌬16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-B⌬16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of ϳ85% SfNIC-B plus ϳ15% SfNIC-B⌬16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNICB⌬pifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1-and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.

Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Mixtures of Complete andpif1- andpif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

Clavijo

Williams

Simón

et al. 2009

“…In addition, ORF5 and ORF2 are homologues of baculovirus ODV envelope protein-encoding gene odv-e56 (5) and of vp91, encoding a baculovirus protein of 91 kDa present in both the capsid and the envelope surrounding the capsid of ODVs (45). Baculovirus P74 and PIF-1 are present only in the ODV envelope (9,14,31). Even though PIF-2 and PIF- 3 have not yet been localized, it is conceivable that these functionally con- served proteins are also present in the ODV envelope.…”

Section: Resultsmentioning

confidence: 99%

The Genome of Gryllus bimaculatus Nudivirus Indicates an Ancient Diversification of Baculovirus-Related Nonoccluded Nudiviruses of Insects

Wang

Kleespies²,

Huger³

et al. 2007

The Gryllus bimaculatus nudivirus (GbNV) infects nymphs and adults of the cricket Gryllus bimaculatus (Orthoptera: Gryllidae). GbNV and other nudiviruses such as Heliothis zea nudivirus 1 (HzNV-1) and Oryctes rhinoceros nudivirus (OrNV) were previously called "nonoccluded baculoviruses" as they share some similar structural, genomic, and replication aspects with members of the family Baculoviridae. Their relationships to each other and to baculoviruses are elucidated by the sequence of the complete genome of GbNV, which is 96,944 bp, has an AT content of 72%, and potentially contains 98 predicted protein-coding open reading frames (ORFs). Forty-one ORFs of GbNV share sequence similarities with ORFs found in OrNV, HzNV-1, baculoviruses, and bacteria. Most notably, 15 GbNV ORFs are homologous to the baculovirus core genes, which are associated with transcription (lef-8, lef-9, lef-4, vlf-1, and lef-5), replication (dnapol), structural proteins (p74, pif-1, pif-2, pif-3, vp91, and odv-e56), and proteins of unknown function (38K, ac81, and 19kda). Homologues to these baculovirus core genes have been predicted in HzNV-1 as well. Six GbNV ORFs are homologous to nonconserved baculovirus genes dnaligase, helicase 2, rr1, rr2, iap-3, and desmoplakin. However, the remaining 57 ORFs revealed no homology or poor similarities to the current gene databases. No homologous repeat (hr) sequences but fourteen short direct repeat (dr) regions were detected in the GbNV genome. Gene content and sequence similarity suggest that the nudiviruses GbNV, HzNV-1, and OrNV form a monophyletic group of nonoccluded double-stranded DNA viruses, which separated from the baculovirus lineage before this radiated into dipteran-, hymenopteran-, and lepidopteran-specific clades of occluded nucleopolyhedroviruses and granuloviruses. The accumulated information on the GbNV genome suggests that nudiviruses form a highly diverse and phylogenetically ancient sister group of the baculoviruses, which have evolved in a variety of highly divergent host orders.

“…Each gene in such cluster is transcribed from its respective promoter, resulting in a series of overlapping coterminal transcripts. In most cases, the overlapping transcripts generally share either a common 5Ј end (7,31) or a coterminal 3Ј end (1,12,13,21,27,28). Transcription of the distal upstream gene may interfere with the transcriptional initiation of the downstream gene via a regulatory mechanism called promoter occlusion (1,11,12,31).…”

Section: Discussionmentioning

confidence: 99%

Autographa californica Multiple Nucleopolyhedrovirus ac76 Is Involved in Intranuclear Microvesicle Formation

Yuan

et al. 2010

In this study, we characterized Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf76 (ac76), which is a highly conserved gene of unknown function in lepidopteran baculoviruses. Transcriptional analysis of ac76 revealed that transcription of multiple overlapping multicistronic transcripts initiates from a canonical TAAG late-transcription start motif but terminates at different 3 ends at 24 h postinfection in AcMNPV-infected Sf9 cells. To investigate the role of ac76 in the baculovirus life cycle, an ac76-knockout virus was constructed using an AcMNPV bacmid system. Microscopy, titration assays, and Western blot analysis demonstrated that the resulting ac76-knockout virus was unable to produce budded viruses. Quantitative real-time PCR analysis demonstrated that ac76 deletion did not affect viral DNA synthesis. Electron microscopy showed that virus-induced intranuclear microvesicles as well as occlusionderived virions were never observed in cells transfected with the ac76-knockout virus. Confocal microscopy analysis revealed that Ac76 was predominantly localized to the ring zone of nuclei during the late phase of infection. This suggests that ac76 plays a role in intranuclear microvesicle formation. To the best of our knowledge, this is the first baculovirus gene identified to be involved in intranuclear microvesicle formation.Baculoviruses are arthropod-specific, rod-shaped, enveloped viruses with circular, supercoiled double-stranded DNA genomes that replicate in the nuclei of host cells (38). The Baculoviridae are divided into four genera: Alphabaculovirus (lepidopteran-specific nucleopolyhedrovirus [NPV]), Betabaculovirus (lepidopteran-specific granuloviruses), Gammabaculovirus (hymenopteran-specific NPV), and Deltabaculovirus (dipteran-specific NPV) (38). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the archetype species of Alphabaculovirus (38). During the AcMNPV life cycle, two virion phenotypes, budded virus (BV) and occlusion-derived virus (ODV), are produced. Both types of virions have similar nucleocapsid structures and identical genetic information, but they differ in the origin and composition of their envelopes and in the roles they play in the baculovirus life cycle (32). During the early phase of infection, viral DNA replicates in the virogenic stroma, and newly synthesized viral genomes are condensed and packaged into rodshaped capsids to form nucleocapsids. Nucleocapsids are transported into the cytoplasm and become enveloped by budding through the GP64-modified plasma membrane, thereby forming BVs. BVs are responsible for spreading infection between susceptible insect tissues and between cells in cell culture. During the late phase of infection, nucleocapsids remain localized within a peristromal ring zone, where they are enveloped in intranuclear microvesicles (whose formation is induced by viral infection) to form ODVs. The ODVs are subsequently embedded into a paracrystalline protein matrix to form occlusion bodies (OBs). ODVs play a role in the horizontal ...