Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia

Abstract

Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor β–induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model.

Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia

“…The ovine cell line used in this study was originally isolated, immortalized, cloned, and characterized in previous studies (Munoz-Gutierrez et al, 2016; Munoz-Gutierrez et al, 2015), which were approved by the Institutional Animal Care and Use Committees of Washington State University (ASAF04575). For these experiments, five clones (434, 438, 439, 440, and 441) previously generated from human telomerase (hTERT) immortalized ovine microglia subline H were utilized (Munoz-Gutierrez et al, 2015).…”

“…The transcripts of 11 genes were quantified by real-time PCR using the CFX96 detection system (Bio-Rad, Hercules, CA, USA) in 20 μl reactions containing 10 μl of SsoAdvanced ™ Universal SYBR ® Green Supermix (Bio-Rad), 0.2 μM of each gene-specific primer, and 2 μl of cDNA diluted 1:100 into water. All primers used in this study were described previously (Budhia et al, 2006; Munoz-Gutierrez et al, 2016; Stanton et al, 2012), except those for NCKAP1L , and are listed in Table 1. NCKAP1L primers were designed based on the Ovis aries NCK-associated protein 1-like mRNA reference sequence (XM_004006271.3) using the primer-blast tool (Ye et al, 2012).…”

“…In an initial study comparing two clones of this subline (Munoz-Gutierrez et al, 2016), we demonstrated that differential permissiveness was not due to differences in PrP C expression and, by analysis of the transcriptome, revealed an association with differential regulation of numerous genes, including those with roles specific to heparin binding (e.g., SEPP1 ), cell proliferation (e.g., RARRES1 ), protein degradation (e.g., SQSTM1 ), and the ECM (e.g., MMP14 ).…”

“…Permissibility was measured using the Standard Scrapie Cell Assay (SSCA) and tested for correlation with cell proliferation rate, MMP2 activity, and transcript levels from a selected subset of genes previously correlated with permissiveness to prions (Munoz-Gutierrez et al, 2016). …”

Immortalized sheep microglial cells are permissive to a diverse range of ruminant viruses

“…Furthermore, two clones derived from subline H have transcriptomes consistent with microglia (Muñoz-Guti errez et al 2016). BT cells and goat synovial membrane (GSM) cells were used for comparison to the sheep microglial.…”