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The folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (NP) vaccines in a timely and reproducible manner. Despite significant advances in in silico design and structure-based assembly, most engineered NPs are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. The failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. Capitalizing on a novel function of RNA as a molecular chaperone (chaperna: chaperone + RNA), we provide a robust protein-folding vehicle that may be implemented to NP assembly in bacterial hosts. The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. Site-specific proteolytic removal of the RID prompted the assemblage of monomers into NPs, which was confirmed by electron microscopy and dynamic light scattering. The mutations that affected the RNA binding to RBD significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of NPs of a defined size. This underscored the RNA-antigen interactions during NP assembly. The sera after mouse immunization effectively interfered with the binding of MERS-CoV RBD to the cellular receptor hDPP4. The results suggest that RNA-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of NPs into highly regular and immunologically relevant conformations. The concentration of the ion Fe2+, salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections.

Interferons (IFNs) mediate direct antiviral activity. They play a crucial role in the early host immune response against viral infections. However, IFN therapy for HBV infection is less effective than for other viral infections.We explored the cellular targets of HBV in response to IFNs using proteome-wide screening.Using LC-MS/MS, we identified proteins downregulated and upregulated by IFN treatment in HBV X protein (HBx)-stable and control cells. We found several IFN-stimulated genes downregulated by HBx, includingWe verified our findings using a mouse model, primary human hepatocytes and human liver tissues. Our data elucidate a mechanism by which HBV evades the host innate immune system.

Zinc is an important trace element in the body and is involved in both the proliferation and growth arrest of many kinds of cells including colorectal epithelial cells. The aim of this study was to identify the molecular mechanism of the growth regulation of colorectal cancer cells by extracellular zinc. Zinc-stimulated activation of the mitogen-activated protein kinase (MAPK) cascade was measured by immunoblotting and Elk-1 dependent trans-reporter gene expression, and zinc-stimulated p21(Cip/WAF1) activation by immunoblotting, Northern blot analysis and immunochemistry. Cell proliferation was measured by thymidine and bromodeoxyuridine (BrdU) incorporation. By treating colorectal cancer cells with 100 microM ZnCl2, MAPKs were activated in two different phases, the initial weak activation occurred within 5 min and this was followed by a stronger and more prolonged activation. Zinc concomitantly activated Raf-1-MEK-MAPK kinases, and induced Elk-1 dependent trans-reporter gene expression. Prolonged activation of MAPKs by 100 microM of ZnCl2 resulted in the induction and nuclear localization of p21(Cip/WAF1) and was related to the inhibition of both thymidine and BrdU incorporations. These results not only suggest the presence of a mechanism for p21(Cip/WAF1) dependent negative regulation of colorectal cancer cell growth by zinc but also suggest potential usage of zinc to control the growth of colorectal cancer cells.