Removal of transposon target sites from the Autographa californica multiple nucleopolyhedrovirus fp25k gene delays, but does not prevent, accumulation of the few polyhedra phenotype

Abstract

Low-cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is seriously hindered by the accumulation of AcMNPV mutants. Specifically, few-polyhedra (FP) mutants, with a reduced yield of occluded virus (polyhedra) and decreased infectivity, usually accumulate upon passaging in cell culture. FP mutations result from transposon insertions in the baculovirus fp25k gene, leading to significantly reduced levels of FP25K protein synthesis. This study evaluated the effects of removing the transposon insertion sites from the wild-type baculovirus fp25k gene; the mutated virus was denoted Ac-FPm. Specifically, this study involved a detailed comparison of wild-type (WT) AcMNPV and Ac-FPm with regard to the proportion of cells having polyhedra, number of polyhedra per cell, the fraction of empty polyhedra, number of occlusion-derived viruses per polyhedron, number of nucleocapsids in the nuclei, FP25K protein synthesis and genetic analysis of the fp25k gene. Removal of TTAA transposon insertion sites from the fp25k gene stabilized FP25K protein synthesis and delayed the appearance of the FP phenotype from passage 5 to passage 10. Electron micrographs revealed that more virus particles were found inside the nuclei of cells infected with Ac-FPm than in the nuclei of cells infected with WT AcMNPV (at passage 10). Abnormalities, however, were observed in envelopment of nucleocapsids and virus particle occlusion within Ac-FPm polyhedra. Thus, the FP phenotype appeared in spite of continued FP25K protein synthesis, suggesting that mechanisms other than fp25k gene disruption can lead to the FP phenotype.

Baculovirus FP25K Localization: Role of the Coiled-Coil Domain

“…Alphabaculovirus FP25Ks are thought to be partially responsible for the general switch between the production of BV and the production of ODV (14)(15)(16). In addition, infection with alphabaculovirus fp25k deletion mutants leads to the accumulation of BV envelope proteins like GP64 (17).…”

“…The amino acid sequences of wild-type FP25K and both the D22P and L36P mutants were entered into the COILS server (http://www.ch.embnet.org/software/COILS_form.html), and the coiled-coil structure of these proteins was predicted (37). The output for this prediction is represented by graphical plots, with all residue-scanning windows (14,21,28) containing the probability of a coiledcoil structure based on similarity to structures in a database of known coiled-coil proteins. High-probability peaks in these plots indicate the likelihood of a coiled-coil structure in that region.…”

“…Since it is not essential in vitro, and inactivation provides a selective advantage, the fp25k gene of baculoviruses readily undergoes mutation by transposition or replication slippage errors resulting in a base insertion or deletion (indel) during infection in cell culture (14,(20)(21)(22). These mutations lead to an evident fewpolyhedra (FP) phenotype, characterized by decreased production of OBs, fewer ODV envelope proteins localized to the nucleus, and increased BV titers (18,(22)(23)(24)(25).…”

“…Unlike other baculoviruses, the occlusion process of betabaculoviruses occurs after the breakdown of the nuclear envelope in a cytoplasmic-nuclear mix (3,12,13). The process of INM blebbing, facilitated by FP25K and other ODV envelope proteins, is thought to be a general switch from BV to ODV production (14)(15)(16). This multifunctional FP25K protein is not required for infection in vitro, but it is necessary in vivo for proper ODV envelopment, virion occlusion, and insect per os infectivity (10,(16)(17)(18)(19).…”

Statistical modeling of cell‐to‐cell variability in viral infection during passaging in suspension cell culture: Application in Monte‐Carlo simulation

“…Fp25k is a late cytoplasmic protein responsible for proper assembly and formation of polyhedra and acts as a switch from exporting BV to maintaining the virus within the cell, that is, ODV formation Saxena et al (2018). It has also been shown that a genetically modified virus with modified transposon insertion sites in the fp25k gene is capable of delaying the formation of FP mutant and defective interfering particle accumulation (Cheng et al, 2013; Giri et al, 2010). None of these studies, however, present a statistical model describing the structural variations present in infected cells, for example, the wide‐ranging polyhedra characteristics, ODV structures, and nucleocapsid packaging in ODVs, that result in a variety of phenotypes.…”