Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively and found accessory protein 8b could enhance viral replication in vivo and in vitro, and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection.
Project description:Background: Lignocellulosic biomass is a promising renewable feedstock for the microbial production of fuels. To release the major fermentable sugars such as glucose and xylose, pretreatment, hydrolysis, and subsequent conditioning of biomass feedstock are needed. During this process, many toxic compounds are produced or introduced which subsequently inhibit microbial growth and in many cases the production titer and rate. An understanding of the toxic effects of compounds found in hydrolysate on the fermentation microorganism is critical to improving biofuel yields in the process. One of the inhibitory compounds is furfural, liberated from hemicelluloses, which strongly inhibits the cell growth and ethanol production especially from xylose. Zymomonas mobilis is a capable ethanologenic bacterium with high ethanol productivity and high levels of ethanol tolerance. The development of robust biocatalyst to tolerate the lignocellulosic pretreatment inhibitors is one of the key elements for economic biofuel production. Results: In this study, the molecular responses of Z. mobilis to furfural, one major pretreatment inhibitor, were investigated using transcriptomic approaches of chip-based microarray. Furfural shock time course experiment with 3 g/L furfural supplemented when cells reach exponential phase and stress response experiment in the presence of 2 g/L furfural from the beginning of fermentation were carried out to study the short and long-term effect of furfural on 8b physiological and transcriptional profiles. The presence and supplementation of furfural negatively affect 8b growth in terms of final biomass and the fermentation time. Transcriptomic studies indicated that the response of 8b to furfural is dynamic, complex and differences exist between short-term shock response and long-term stress response. However, the gene function categories are similar with most downregulated genes related to translation and biosynthesis, while the furfural-upregulated genes were mostly related to cellular processes of general stress response and energy metabolism. Conclusions: Similar to previous report that acetate inhibited the growth of Z. mobilis 8b in RM using glucose or xylose as carbon source, the existence or supplementation of another major hydrolysate inhibitor furfural also inhibited 8b growth with slowing the substrate utilization and ethanol production. The difference between carbon sources is more dramatic than that of the major hydrolysate inhibitors of both NH4OAc (GSE57553) and furfural (this study). Several gene targets have been selected for genetic studies with promising preliminary results.