Biology subjects

Pruijssers, A. J.

Publications and source records attributed to Pruijssers, A. J..

4 recordsLinked to original sources

bioRxiv2022

Distinct genetic determinants and mechanisms of SARS-CoV-2 resistance to remdesivir

The nucleoside analog remdesivir (RDV) is an FDA-approved antiviral for the treatment of SARS- CoV-2 infections, and as such it is critical to understand potential genetic determinants and barriers to RDV resistance. In this study, SARS-CoV-2 was subjected to 13 passages in cell culture with increasing concentrations of GS-441524, the parent nucleoside of RDV. At passage 13 the RDV resistance of the lineages ranged from 2.7-to 10.4-fold increase in EC50. Sequence analysis of the three lineage populations identified non-synonymous mutations in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I and C799F/R. Two of the three lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first then combined with S759A. Introduction of the S759A and V792I substitutions at homologous nsp12 positions in viable isogenic clones of the betacoronavirus murine hepatitis virus (MHV) demonstrated their transferability across CoVs, up to 38-fold RDV resistance in combination, and a significant replication defect associated with their introduction. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a [~]10- fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, while nsp12-V792I diminished the UTP concentration needed to overcome the template-dependent inhibition associated with RDV. The in vitro selected substitutions here identified were rare or not detected in the >6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in various clinical settings. One Sentence SummarySARS-CoV-2 develops in vitro resistance to remdesivir by distinct and complementary mutations and mechanisms in the viral polymerase

microbiology↗

bioRxiv2020

Neuron-intrinsic NF-κB Signaling Mediates Reovirus Virulence

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germline ablation of NF-{kappa}B subunit p50. It is not known whether the pro-apoptotic function of NF-{kappa}B is mediated by neuron-intrinsic processes, NF-{kappa}B-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-{kappa}B signaling in reovirus-induced neuronal injury, we established mice that lack NF-{kappa}B p65 expression in neurons using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural NF-{kappa}B p65 (Nsp65-/-) mice survived. While viral loads in brains of WT and Nsp65-/- were comparable, histological analysis revealed that reovirus antigen-positive areas in the brain of WT mice displayed enhanced cleaved caspase-3 immunoreactivity, a marker of apoptosis, compared with Nsp65-/- mice. These data suggest that neuron-intrinsic NF-{kappa}B-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected cortices of WT and Nsp65-/- mice suggests that NF-{kappa}B activation in neurons upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-{kappa}B-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations

microbiology↗

bioRxiv2020

Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 M). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 M) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.

microbiology↗

bioRxiv2020

The coronavirus proofreading exoribonuclease mediates extensive viral recombination

Coronaviruses (CoVs) emerge as zoonoses and cause severe disease in humans, demonstrated by the SARS-CoV-2 (COVID-19) pandemic. RNA recombination is required during normal CoV replication for subgenomic mRNA (sgmRNA) synthesis and generates defective viral genomes (DVGs) of unknown function. However, the determinants and patterns of CoV recombination are unknown. Here, we show that divergent {beta}-CoVs SARS-CoV-2, MERS-CoV, and murine hepatitis virus (MHV) perform extensive RNA recombination in culture, generating similar patterns of recombination junctions and diverse populations of DVGs and sgmRNAs. We demonstrate that the CoV proofreading nonstructural protein (nsp14) 3-to-5 exoribonuclease (nsp14-ExoN) is required for normal CoV recombination and that its genetic inactivation causes significantly decreased frequency and altered patterns of recombination in both infected cells and released virions. Thus, nsp14-ExoN is a key determinant of both high fidelity CoV replication and recombination, and thereby represents a highly-conserved and vulnerable target for virus inhibition and attenuation.

microbiology↗