No abstract

Baculoviruses are insect viruses extensively exploited as eukaryotic protein expression vectors. Molecular biology studies have provided exciting discoveries on virus–host interactions, but the application of omic high-throughput techniques on the baculovirus–insect cell system has been hampered by the lack of host genome sequencing. While a broader, systems-level analysis of biological responses to infection is urgently needed, recent advances on proteomic studies have yielded new insights on the impact of infection on the host cell. These works are reviewed and critically assessed in the light of current biological knowledge of the molecular biology of baculoviruses and insect cells.

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single‐cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell‐intrinsic correlation between glycolytic flux and levels of fructose‐1,6‐bisphosphate (FBP), we transplanted the B. subtilis FBP‐binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms 1 1 2 The recent approval of vaccines and gene therapy products for human use produced in the 38 Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and 39 versatility of this platform. The interest in developing robust production processes emerges to 40 cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, 41 TARGETED SUPPLEMENTATION DESIGN FOR IMPROVED PRODUCTION AND 3 QUALITY OF ENVELOPED VIRAL PARTICLES IN INSECT CELL-BACULOVIRUSwe addressed the impact of the baculovirus infection on the physiology of insect host cell lines, 42 identifying key cellular pathways enrolled in heterologous gene/protein expression. In the 43 present work, this knowledge was applied to design tailored media supplementation schemes to 44 boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-45 VLP) and baculovirus vectors (BV). 46The addition of reduced glutathione, antioxidants and polyamines increased the cell 47 specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most 48 critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-49 VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol 50 supplementation improved baculovirus stock quality, by preventing the accumulation of non-51 infectious particles during viral replication while selectively increasing infectious particles 52 production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, 53were also increased. 54The correlation between supplement addition and systems productivity was extensively 55 analyzed, providing a critical assessment on final product quantity and quality as drivers of 56 bioprocess optimization efforts. 57 58