Gene Expression Analysis of Adapted Insect Cells during Influenza VLP Production Using RNA-Sequencing

Abstract

Adaptive laboratory evolution has been used to improve production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) in insect cells. However, little is known about the underlying biological mechanisms promoting higher HA-VLP expression in such adapted cell lines. In this article, we present a study of gene expression patterns associated with high-producer insect High Five cells adapted to neutral pH, in comparison to non-adapted cells, during expression of influenza HA-VLPs. RNA-seq shows a decrease in the amount of reads mapping to host cell genomes along infection, and an increase in those mapping to baculovirus and transgenes. A total of 1742 host cell genes were found differentially expressed between adapted and non-adapted cells throughout infection, 474 of those being either up- or down-regulated at both time points evaluated (12 and 24 h post-infection). Interestingly, while host cell genes were found up- and down-regulated in an approximately 1:1 ratio, all differentially expressed baculovirus genes were found to be down-regulated in infected adapted cells. Pathway analysis of differentially expressed genes revealed enrichment of ribosome biosynthesis and carbohydrate, amino acid, and lipid metabolism. In addition, oxidative phosphorylation and protein folding, sorting and degradation pathways were also found to be overrepresented. These findings contribute to our knowledge of biological mechanisms of insect cells during baculovirus-mediated transient expression and will assist the identification of potential engineering targets to increase recombinant protein production in the future.

Dissecting insect cell heterogeneity during influenza VLP production using single-cell transcriptomics

“…M1 and HA proteins were identified by Western blot ( Figure 1B ), and particles resembling influenza HA-VLPs, both in size and morphology, detected by TEM ( Figure 1C ). These results are in line with those previously reported ( Silvano et al, 2022 ) and demonstrate that HA-VLPs were successfully produced.…”

“…Overall, 921 host cell genes were found to be significantly (q value <.05) associated with pseudotime. Pathway enrichment analysis allowed to identify biological processes varying most along infection, of which those associated to translation machinery, energy metabolism, protein folding and endocytosis ( Figure 4B ) are some examples, in good agreement to what we have previously found in bulk RNA sequencing analysis ( Silvano et al, 2022 ; Virgolini et al, 2022 ). The relative expression of a selected number of genes involved in these pathways is presented in Supplementary Figure S3 , illustrating the significant transcriptional changes in cells upon infection.…”

“…Overall, the enrichment analysis allowed to identify several pathways (e.g., ribosome, spliceosome, oxidative phosphorylation) that were common to those previously identified in our bulk RNA sequencing study ( Silvano et al, 2022 ), demonstrating the robustness and replicability of the data. Importantly, single-cell RNA sequencing allowed to evaluate single cells at different states of infection within the same sample and capture the transcriptional changes associated with the infection process (not possible with the bulk RNA sequencing approach), thus elevating the importance of single-cell omics analysis in the IC-BEVS system.…”

“…For example, RNA-seq has been used to assess the transcriptional changes of alphanodavirus-free High Five cells upon infection by Autographa californica multiple nucleopolyhedrovirus ( Ac MNPV) ( Chen et al, 2013 ; Chen et al, 2014 ), providing a global picture of the AcMNPV transcription regulation throughout the infection cycle. As the knowledge of the IC-BEVS grows, potential engineering targets to increase recombinant protein production are being identified ( Silvano et al, 2022 ).…”

“…To date, the understanding of the IC-BEVS transcriptome has been mostly relying on bulk RNA-seq analysis ( Nguyen et al, 2013 ; Chen et al, 2014 ; Silvano et al, 2022 ; Virgolini et al, 2022 ). For instance, we have previously assessed whole transcriptome changes in High Five insect cells during expression of influenza HA-displaying virus-like particles (HA-VLPs) using IC-BEVS, which enabled to identify key biological processes impacted by virus infection ( Silvano et al, 2022 ). Although these studies uncover transcriptional changes in insect cell response to baculovirus infection, they only provide rough models of the host cell response.…”