Cells are thought to adapt to proteasome inhibition by using alternative pathways for degradation such as autophagy. Sha et al. now report that cells rapidly induce GABARAPL1 and p62 upon proteasome inhibition, but this promotes cell survival by sequestering ubiquitinated and sumoylated proteins long before the cells induce other Atg genes and activate autophagy.

Human norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., genogroup I genotype 1 [GI.1]). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientations of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important cofactors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2′FL). In the current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2′FL enhanced the blocking potential with an additive (Nano-7) or synergistic (Nano-94) effect. We also found that GII Nanobodies with 2′FL also enhanced inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas 2′FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2′FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup. IMPORTANCE The discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known cofactor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., the addition of Nano-7 or Nano-94 with 2′FL, the effect of inhibition was increased. Therefore, combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse and are continually changing the capsid landscape, and few conserved epitopes have so far been identified.

Norovirus infection is the major cause of nonbacterial gastroenteritis in humans and has been the subject of numerous studies investigating the virus's biophysical properties and biochemical function with the aim of deriving novel and highly potent entry inhibitors to prevent infection. Recently, it has been shown that the protruding P domain dimer (P-dimer) of a GII.10 Norovirus strain exhibits two new binding sites for l-fucose in addition to the canonical binding sites. Thus, these sites provide a novel target for the design of multivalent fucose ligands as entry inhibitors of norovirus infections. In this current study, a first generation of multivalent fucose-functionalized glycomacromolecules was synthesized and applied as model structures to investigate the potential targeting of fucose binding sites in human norovirus P-dimer. Following previously established solid phase polymer synthesis, eight precision glycomacromolecules varying in number and position of fucose ligands along an oligo(amidoamine) backbone were obtained and then used in a series of binding studies applying native MS, NMR, and X-ray crystallography. We observed only one fucose per glycomacromolecule binding to one P-dimer resulting in similar binding affinities for all fucose-functionalized glycomacromolecules, which based on our current findings we attribute to the overall size of macromolecular ligands and possibly to steric hindrance.

Human noroviruses are highly contagious and a major problem in closed institutions, such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics.

Norovirus is the most important cause of acute gastroenteritis, yet there are still no antivirals, vaccines, or treatments available. Several studies have shown that norovirus-specific monoclonal antibodies, Nanobodies, and natural extracts might function as inhibitors. Therefore, the objective of this study was to determine the antiviral potential of additional natural extracts, honeys, and propolis samples. Norovirus GII.4 and GII.10 virus-like particles (VLPs) were treated with different natural samples and analyzed for their ability to block VLP binding to histo-blood group antigens (HBGAs), which are important norovirus co-factors. Of the 21 natural samples screened, date syrup and one propolis sample showed promising blocking potential. Dynamic light scattering indicated that VLPs treated with the date syrup and propolis caused particle aggregation, which was confirmed using electron microscopy. Several honey samples also showed weaker HBGA blocking potential. Taken together, our results found that natural samples might function as norovirus inhibitors.

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