Turnbaugh, E. | Search bioRxiv

bioRxiv2026

Fluorescent non-canonical amino acid as a site-specific conformational probe of prion formation

The pathogenic conversion of the cellular prion protein (PrPC) into the {beta}-sheet-rich isoform PrPSc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive non-canonical amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and non-infectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole (BV) PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new and broadly applicable strategy for studying conformational dynamics in amyloid-forming proteins.

biochemistry↗

bioRxiv2024

Asymptomatic neonatal herpes simplex virus infection in mice leads to long-term cognitive impairment

Neonatal herpes simplex virus (nHSV) is a devastating infection impacting approximately 14,000 newborns globally each year. Infection is associated with high neurologic morbidity and mortality, making early intervention and treatment critical. Clinical outcomes of symptomatic nHSV infections are well-studied, but little is known about the frequency of, or outcomes following, sub-clinical or asymptomatic nHSV. Given the ubiquitous nature of HSV infection and frequency of asymptomatic shedding in adults, subclinical infections are underreported, yet could contribute to long-term neurological damage. To assess potential neurological morbidity associated with subclinical nHSV infection, we developed a low-dose (100 PFU) HSV infection protocol in neonatal C57BL/6 mice. At this dose, HSV DNA was detected in the brain by PCR but was not associated with acute clinical symptoms. However, months after initial inoculation with 100 PFU of HSV, we observed impaired mouse performance on a range of cognitive and memory performance tasks. Memory impairment was induced by infection with either HSV-1 or HSV-2 wild-type viruses, but not by a viral mutant lacking the autophagy-modulating Beclin-binding domain of the neurovirulence gene {gamma}34.5. Retroviral expression of wild type {gamma}34.5 gene led to behavioral pathology in mice, suggesting that {gamma}34.5 expression may be sufficient to cause cognitive impairment. Maternal immunization and HSV-specific antibody treatment prevented offspring from developing neurological sequelae following nHSV-1 infection. Altogether, these results support the idea that subclinical neonatal infections may lead to cognitive decline in adulthood, with possible profound implications for research on human neurodegenerative disorders such as Alzheimers Disease.

microbiology↗