A Single Amino Acid Alteration in the Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Glycoprotein Confers Resistance to the Inhibitory Effects of Zanamivir on Receptor Binding and Neuraminidase Activity

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-GU-DANA; zanamivir), a sialic acid transition-state analog designed to fit the influenza virus neuraminidase catalytic site, possesses antiviral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity. In the present study, a 4-GU-DANA-resistant HPF3 virus variant (ZM1) was generated by serial passage in the presence of 4-GU-DANA. ZM1 exhibited a markedly fusogenic plaque morphology and harbored two HN gene mutations resulting in two amino acid alterations, T193I and I567V. Another HPF3 variant studied in parallel, C-0, shared an alteration at T193 and exhibited similar plaque morphology but was not resistant to 4-GU-DANA. Neuraminidase assays revealed a 15-fold reduction in 4-GU-DANA sensitivity for ZM1 relative to the wild type (WT) and C-0. The ability of ZM1 to bind sialic acid receptors was inhibited 10-fold less than for both WT and C-0 in the presence of 1 mM 4-GU-DANA. ZM1 also retained infectivity at 15-fold-higher concentrations of 4-GU-DANA than WT and C-0. A single amino acid alteration at HN residue 567 confers these 4-GU-DANA-resistant properties. An understanding of ZM1 and other escape variants provides insight into the effects of this small molecule on HN function as well as the role of the HN glycoprotein in HPF3 pathogenesis

Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress–responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D