Human coronaviruses (HCoV) are recognized respiratory pathogens that may be involved in other pathologies such as central nervous system (CNS) diseases. To investigate whether leukocytes could participate in respiratory pathologies and serve as vector for viral spread towards other tissues, the susceptibility of human leukocytic cell lines and peripheral blood mononuclear cells (PBMC) to HCoV-229E and HCoV-OC43 infection was investigated. Human primary monocytes/macrophages were susceptible to HCoV-229E infection, but strongly restricted HCoV-OC43 replication. Moreover, productive HCoV-229E infection of primary monocytes and of the THP-1 monocytic cell line led to their activation, as indicated by the production of pro-inflammatory mediators, including TNF-alpha, CCL5, CXCL10 and CXCL11 and MMP-9. Moreover, an in vitro chemotaxis assay showed that motility towards chemokines of THP-1 cells and primary monocytes was increased following an acute or persistent HCoV-229E infection. Taken together, these results suggest that infected monocytes could serve as a reservoir for HCoV-229E, become activated, participate in the exacerbation of pulmonary pathologies, as well as serve as potential vectors for viral dissemination to host tissues, where it could be associated with other pathologies.

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Protein design is currently used for the creation of new proteins with desirable traits. In our lab, we focus on the synthesis of proteins with high essential amino acid content, having potential application in animal nutrition. One of the limitations we face in this endeavor is the achievement of stable proteins in spite of a highly biased amino acid content. We report here the synthesis and characterization of MB-1Trp, a protein with a tailored content in selected essential amino acids. The protein is a Tyr62-Trp mutant of the parent molecule MB-1 described earlier. The new protein is largely helical as per design, is well folded, and has a melting temperature of 55³C. Its resistance to proteolytic degradation compares to that of cytochrome c, a protein of similar size. Design strategy used for MB-1Trp is discussed with regards to its applicability toward the creation of efficient nutritional proteins. ß

No abstract

Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.