Rapid Titer Determination of Baculovirus by Quantitative Real-Time Polymerase Chain Reaction

ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5= rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment. IMPORTANCEBaculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles. The Baculoviridae are a family of insect-specific doublestranded DNA (dsDNA) viruses. Viruses from this family are characterized by rod-shaped, enveloped nucleocapsids with circular, covalently closed, double-stranded DNA genomes of 80 to 180 kbp (1-3). The Baculoviridae family comprises four genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus. Alphabaculoviruses and betabaculoviruses infect lepidopteran larvae, whereas gammabaculoviruses and deltabaculoviruses infect hymenopteran and dipteran larvae, respectively (4). The alphabaculoviruses are further divided into group I and group II on the basis of phylogenetic analysis (5) and the type of envelope fusion protein (GP64 and F, respectively) (6).The infection cycle of baculoviruses includes two distinct viral phenotypes: budded virus (BV) and occlusion-derived virus (ODV). Both BVs and ODVs are identical in terms of nucleocapsid structure and genetic information, but the composition of their envelopes is different to accommodate their respective functions in the infection cycle (7). ODVs, which become embedded ...

Section: Methodsmentioning

confidence: 99%

Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

Tao

Choi

Kim

et al. 2015

“…Cells were cultured at 26°C in TC100 insect medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS). Virus titers were determined by quantitative PCR (25).…”

Section: Methodsmentioning

confidence: 99%

RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells

Liu

Wang

Hsiao

et al. 2009

Self Cite

In recent years, baculovirus has emerged as a tool for high-efficiency gene transfer into mammalian cells. However, the level of gene expression is often limited by the strength of the mammalian promoter used. Here, we show that the baculovirus RING protein IE2 is a strong, promiscuous trans-activator in mammalian cells, dramatically upregulating the cytomegalovirus (CMV) promoter in both Vero E6 and U-2OS cells. Further study of the cellular mechanism for the activation led to the discovery of a novel IE2 nuclear body structure which contains a high concentration of G-actin and closely associates with RNA polymerase II, PML, and SUMO1. IE2 mutagenesis studies indicated that the RING and coiled-coil domains of IE2 were necessary for nuclear body formation, as well as for strong activation of the CMV promoter in mammalian cells. Overall, this study shows that the IE2 trans-activator could significantly advance the use of baculovirus in mammalian gene transfer and protein production.

“…To further detect whether there was noninfectious progeny BVs produced in ac93KO, quantitative real-time PCR (qPCR) was performed to titrate the baculovirus stocks, as previously described (23,26). The qPCR product corresponded to a 100-bp region of the AcMNPV essential gene, gp41 (36).…”

Section: Cells and Virusesmentioning

confidence: 99%

Identification of Autographa californica Nucleopolyhedrovirus ac93 as a Core Gene and Its Requirement for Intranuclear Microvesicle Formation and Nuclear Egress of Nucleocapsids

Yuan

Huang

Wei

et al. 2011

Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusionderived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.