Esko, J. | Search bioRxiv

bioRxiv2024

Global siRNA Screen Reveals Critical Human Host Factors of SARS-CoV-2 Multicycle Replication

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased towards the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published datasets to reveal pathways supported by orthogonal datasets, including transcriptional regulation, epigenetic modifications, and MAPK signalling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 27 proteins that were determined to impact assembly and release. Additionally, a subset of proteins were tested across other coronaviruses revealing 17 potential pan-coronavirus targets. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry, and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

systems biology↗

bioRxiv2023

Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a major extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for HS accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase, N-sulfotransferase), from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prion strains. Live PET imaging revealed rapid clearance of prion protein monomers into the CSF in mice expressing unsulfated HS, further suggesting that HS sulfate groups hinder transit of extracellular prion monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme target to facilitate aggregate clearance. Author summaryPrions cause a rapidly progressive neurologic disease and death with no curative treatment available. Prion aggregates accumulate exponentially in the brain in affected individuals triggering neuronal loss and neuroinflammation. Yet the additional molecules that facilitate aggregation are largely unknown, and their identification may lead to new therapeutic targets. We have found that prions in the brain preferentially bind to a highly sulfated endogenous polysaccharide, known as heparan sulfate (HS). Here we use genetically modified mice that express poorly sulfated neuron-derived HS, and infect mice with different prions strains. We find that the mice infected with a plaque-forming prion strain show a prolonged survival and fewer plaques compared to the controls. We also found that the prion protein was efficiently transported in the interstitial fluid in mice having poorly sulfated HS, suggesting that the prion protein is more readily cleared from the brain. Our study provides insight into how HS retains prion aggregates in the brain to accelerate disease and indicates the specific HS biosynthetic enzymes to target for enhancing protein clearance.

pathology↗