Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k

Giri, Lopamudra; Feiss, Michael; Bonning, Bryony C.; Murhammer, David W.

doi:10.1099/vir.0.036566-0

Cited by 26 publications

(38 citation statements)

References 36 publications

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“…DIPs also decreased the productivity of continuous protein production using the baculovirus insect cell expression system [58,59]. Hence, recombinant baculovirus expression vectors have been constructed to increase the genetic stability and decrease the generation of DIPs [60,61]. Moreover, it has been reported that high yields of recombinant SV40 vectors for gene therapy applications can be achieved reproducibly by quality controls and infection protocols that reduce the risk of DIP accumulation [62].…”

Section: Mode Of Interference Employed By Viral Dipsmentioning

confidence: 99%

Defective interfering viruses and their impact on vaccines and viral vectors

Frensing

2015

Biotechnology Journal

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Defective interfering particles (DIPs) have been found for many important viral pathogens and it is believed that most viruses generate DIPs. This article reviews the current knowledge of the generation and amplification of DIPs, which possess deletions in the viral genome but retain the ability to replicate in the presence of a complete helper virus. In addition, mechanisms are discussed by which DIPs interfere with the replication of their helper virus leading to the production of mainly progeny DIPs by coinfected cells. Even though DIPs cannot replicate on their own, they are biologically active and it is well known that they have a huge impact on virus replication, evolution, and pathogenesis. Moreover, defective genomes are potent inducers of the innate immune response. Yet, little attention has been paid to DIPs in recent years and their impact on biotechnological products such as vaccines and viral vectors remains elusive in most cases. With a focus on influenza virus, this review demonstrates that DIPs are important for basic research on viruses and for the production of viral vaccines and vectors. Reducing the generation and/or amplification of DIPs ensures reproducible results as well as high yields and consistent product quality in virus production.

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Section: Mode Of Interference Employed By Viral Dipsmentioning

confidence: 99%

Defective interfering viruses and their impact on vaccines and viral vectors

Frensing

2015

Biotechnology Journal

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“…The existence of virus variants with large genomic deletions has since been confirmed in many virus families [2], including the Alphabaculoviruses [3,4]. DI viruses are generated almost instantly and accumulate rapidly when baculoviruses are introduced into cultured insect cells [5,6], leading to problems with sustained expression of heterologous proteins [3] in this widely used expression system [7]. DI viruses are thought to replicate much faster than viruses with a full-length genome, due to their smaller genome sizes.…”

Section: Introductionmentioning

confidence: 99%

Complex dynamics of defective interfering baculoviruses during serial passage in insect cells

et al. 2013

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Defective interfering (DI) viruses are thought to cause oscillations in virus levels, known as the 'Von Magnus effect'. Interference by DI viruses has been proposed to underlie these dynamics, although experimental tests of this idea have not been forthcoming. For the baculoviruses, insect viruses commonly used for the expression of heterologous proteins in insect cells, the molecular mechanisms underlying DI generation have been investigated. However, the dynamics of baculovirus populations harboring DIs have not been studied in detail. In order to address this issue, we used quantitative real-time PCR to determine the levels of helper and DI viruses during 50 serial passages of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Sf21 cells. Unexpectedly, the helper and DI viruses changed levels largely in phase, and oscillations were highly irregular, suggesting the presence of chaos. We therefore developed a simple mathematical model of baculovirus-DI dynamics. This theoretical model reproduced patterns qualitatively similar to the experimental data. Although we cannot exclude that experimental variation (noise) plays an important role in generating the observed patterns, the presence of chaos in the model dynamics was confirmed with the computation of the maximal Lyapunov exponent, and a Ruelle-Takens-Newhouse route to chaos was identified at decreasing production of DI viruses, using mutation as a control parameter. Our results contribute to a better understanding of the dynamics of DI baculoviruses, and suggest that changes in virus levels over passages may exhibit chaos.

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“…Thus, DGs need the viral proteins provided by the wild-type virus in the same host cell so that they can replicate and produce the defective interfering particles (DIPs). Like many DNA viruses (53,54), baculovirus generates DGs during serial undiluted passage (55,56), and certain parts of the baculovirus genome are always retained in the DGs (55,57,58). The smallest DG of AcMNPV generated in a previous study (55) consists of three main parts, including the region from gp41 to ac84 (designated in this study retained region 1, or Rr1), the region from gp64 to p10 (designated in this study retained region 2, or Rr2), and the region from pe38 to polh (designated in this study retained region 3, or Rr3), as shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis -Acting Element That Is Essential for Nucleocapsid Assembly

Huang

Pan

Zhu

et al. 2017

J Virol

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Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83-encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans. Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis-acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis-acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis-acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process.IMPORTANCE Virus nucleocapsid assembly usually requires specific cis-acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis-acting elements in the viral genome.

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Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k

Cited by 26 publications

References 36 publications

Defective interfering viruses and their impact on vaccines and viral vectors

Defective interfering viruses and their impact on vaccines and viral vectors

Complex dynamics of defective interfering baculoviruses during serial passage in insect cells

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis -Acting Element That Is Essential for Nucleocapsid Assembly

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