Abstract
Immortalized uninfected septal (SEP) neurons proliferate but after physiological mitotic arrest they express differentiated neuronal characteristics including enhanced cell-to-cell membrane contacts and ≥ 8 fold increases in host prion protein (PrP). We compared proliferating uninfected and Creutzfeldt-Jakob Disease (CJD) agent infected cells with their arrested counterparts over 33 days by quantitative mRNA and protein blot analyses. Surprisingly, uninfected arrested cells increased interferon-β (IFN-β) mRNA by 2.5–8 fold; IFN-β mRNA elevations were not previously associated with neuronal differentiation. SEP cells with high CJD infectivity titers produced a much larger 40–68-fold increase in IFN-β mRNA, a classic host anti-viral response that is virucidal for RNA but not DNA viruses. High titers of CJD agent also induced dramatic decreases in host PrP, a protein needed for productive agent replication. Uninfected arrested cells produced large sustained 20–30-fold increases in PrP mRNA and protein, whereas CJD arrested cells showed only transient small 5-fold increases in PrP. A > 10-fold increase in infectivity, but not PrP misfolding, induced host PrP reductions that can limit CJD agent replication. In contrast to neuronal lineage cells, functionally distinct migratory microglia with high titers of CJD agent do not induce an IFN-β mRNA response. Because they have 1/50th of PrP of an average brain cell, microglia would be unable to produce the many new infectious particles needed to induce a large IFN-β response by host cells. Instead, microglia and related cells can be persistent reservoirs of infection and spread. Phase separations of agent-associated molecules in neurons, microglia and other cell types can yield new insights into the molecular structure, persistent, and evasive behavior of CJD-type agents.
Highlights
Host prion protein (PrP) mRNA transcription sharply increases during brain synapse formation (Lieberburg, 1987; Manson et al, 1992)
Neuronal differentiation features were enhanced by an 8-day arrest, including 8-fold elevations in host prion protein (PrP), an ultrastructural increase in cell-to-cell plasma membrane attachments and formation of many nanotubes from cell to cell (Miyazawa et al, 2010); Neurofilament RNA by Northern blot hybridization has been reported (Eves et al, 1994)
The above data highlight three key changes induced by high titers of FU-Creutzfeldt-Jakob Disease (CJD) agent in re-arrested neuronal SEP cells: (1) enormous elevations of IFN-β RNA, (2) significantly diminished transcription of host PrP mRNA and protein production, and (3) the reappearance of T antigen (Tag) protein in re-arrested highly infectious cells
Summary
Host prion protein (PrP) mRNA transcription sharply increases during brain synapse formation (Lieberburg, 1987; Manson et al, 1992). Encephalopathies (TSEs) as shown by PrP knockout studies in scrapie (Büeler et al, 1993). It is commonly believed that the causal infectious agent in TSEs is a misfolded form of host PrP without any nucleic acid component (Prusiner, 1998). All highly infectious CJD and scrapie preparations contain significant amounts of long nucleic acids when evaluated using contemporary detection techniques (Manuelidis, 2011). Nucleases that destroy nucleic acids but have no effect on PrP, reduce infectivity by ~1,000 fold (Botsios and Manuelidis, 2016). Misfolded PrP forms amyloid fibrils and on Western blots displays truncated proteinase K resistant bands (PrP-res) and nucleic acids, including DNA aptamers, can promote PrP fibrillation as shown in phase separation studies (Matos et al, 2020; do Amaral and Cordeiro, 2021). PrP-res continues to accumulate even after infected cells have lost all of their infectivity (Miyazawa et al, 2012)
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