a b s t r a c tThe coronavirus (CoV) N protein oligomerizes via its carboxyl terminus. However, the oligomerization mechanism of the C-terminal domains (CTD) of CoV N proteins remains unclear. Based on the protein disorder prediction system, a comprehensive series of HCoV-229E N protein mutants with truncated CTD was generated and systematically investigated by biophysical and biochemical analyses to clarify the role of the C-terminal tail of the HCoV-229E N protein in oligomerization. These results indicate that the last C-terminal tail plays an important role in dimer-dimer association. The C-terminal tail peptide is able to interfere with the oligomerization of the CTD of HCoV-229E N protein and performs the inhibitory effect on viral titre of HCoV-229E. This study may assist the development of anti-viral drugs against HCoV. Structured summary of protein interactions:N and C-terminal tail peptide bind by cosedimentation in solution (View interaction) N and N bind by cosedimentation in solution (View Interaction: 1,2,3,4,5,6,7,8,9,10,11,12) C-terminal tail peptide and N bind by fluorescence technology (View interaction) N and N bind by cross-linking study (View interaction) N and N bind by cross-linking study (View Interaction: 1,2,3,4) Crown

ZnO nanoparticles (ZnO-NPs) are widely used in the engineering and cosmetic industries, and inhaled airborne particles pose a known hazard to human health; their translocation into humans is a recognized public health concern. The pulmonary-blood pathway for ZnO-NP toxicity is well documented, but whether translocation of these particles can also occur via an olfactory bulb-brain route remains unclear. The potential toxicity of ZnO-NPs for the human central nervous system (CNS) is predicated on the possibility of their translocation. Our study investigated translocation of ZnO-NPs both in vitro using the neuronal cell line PC12 and in vivo in a Sprague-Dawley rat model. Our findings indicate that the zinc-binding dye, Newport-Green DCF, binds ZnO stoichiometrically and that ZnO-NP concentration can therefore be measured by the fluorescence intensity of the bound dye in confocal fluorescence microscopy. Confocal data obtained using Newport-Green DCF-2 K(+)-conjugated ZnO-NPs along with the membrane probe FM1-43 demonstrated endocytosis of ZnO-NPs by PC12 cells. In addition, Fluozin-3 measurement showed elevation of cytosolic Zn(2+) concentration in these cells. Following in vivo nasal exposure of rats to airborne ZnO-NPs, olfactory bulbs and brains that were examined by Newport-Green fluorescence and TEM particle measurement clearly showed the presence of ZnO-NPs in brain. We conclude that an olfactory bulb-brain translocation pathway for airborne ZnO-NPs exists in rats, and that endocytosis is required for interneuron translocation of these particles.

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Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane (M) proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles, and of forming virus-like particles (VLPs) when coexpressed with SARS-CoV nucleocapsid (N) protein. According to previous deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail (218LL219) is required for efficient N packaging into VLPs. Results from cross-linking VLP analyses suggest that the cysteine residues 63, 85 and 158 are not in close proximity to the M dimer interface. We noted a significant reduction in M secretion due to serine replacement for C158, but not for C63 or C85. Further analysis suggests that C158 is involved in M-N interaction. In addition to mutations of the highly conserved 107-SWWSFNPE-114 motif, substitutions at codons W19, W57, P58, W91, Y94 or F95 all resulted in significantly reduced VLP yields, largely due to defective M secretion. VLP production was not significantly affected by a tryptophan replacement of Y94 or F95 or a phenylalanine replacement of W19, W57 or W91. Combined, these results indicate the involvement of specific M amino acids during SARS-CoV virus assembly, and suggest that aromatic residue retention at specific positions is critical for M function in terms of directing virus assembly.

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