Specialized cis-Acting RNA Elements Balance Genome Cyclization to Ensure Efficient Replication of Yellow Fever Virus.

Genome cyclization is essential for viral RNA (vRNA) replication of the vertebrate-infecting flaviviruses, and yet its regulatory mechanisms are not fully understood. Yellow fever virus (YFV) is a notorious pathogenic flavivirus. Here, we demonstrated that a group of cis-acting RNA elements in YFV balance genome cyclization to govern efficient vRNA replication. It was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) is conserved in the YFV clade and is important for efficient YFV propagation. By using two different replicon systems, we found that the function of the DCS-HP is determined primarily by its secondary structure and, to a lesser extent, by its base-pair composition. By combining in vitro RNA binding and chemical probing assays, we found that the DCS-HP orchestrates the balance of genome cyclization through two different mechanisms, as follows: the DCS-HP assists the correct folding of the 5' end in a linear vRNA to promote genome cyclization, and it also limits the overstabilization of the circular form through a potential crowding effect, which is influenced by the size and shape of the DCS-HP structure. We also provided evidence that an A-rich sequence downstream of the DCS-HP enhances vRNA replication and contributes to the regulation of genome cyclization. Interestingly, diversified regulatory mechanisms of genome cyclization, involving both the downstream of the 5'-cyclization sequence (CS) and the upstream of the 3'-CS elements, were identified among different subgroups of the mosquito-borne flaviviruses. In summary, our work highlighted how YFV precisely controls the balance of genome cyclization to ensure viral replication. IMPORTANCE Yellow fever virus (YFV), the prototype of the Flavivirus genus, can cause devastating yellow fever disease. Although it is preventable by vaccination, there are still tens of thousands of yellow fever cases per year, and no approved antiviral medicine is available. However, the understandings about the regulatory mechanisms of YFV replication are obscure. In this study, by a combination of bioinformatics, reverse genetics, and biochemical approaches, it was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) promotes efficient YFV replication by modulating the conformational balance of viral RNA. Interestingly, we found specialized combinations for the downstream of the 5'-cyclization sequence (CS) and upstream of the 3'-CS elements in different groups of the mosquito-borne flaviviruses. Moreover, possible evolutionary relationships among the various downstream of the 5'-CS elements were implied. This work highlighted the complexity of RNA-based regulatory mechanisms in the flaviviruses and will facilitate the design of RNA structure-targeted antiviral therapies.

[Effects of endothelial progenitor cells on proliferation and biological function of hepatic stellate cells under shear stress].

OBJECTIVE: To investigate the effects of endothelial progenitor cells (EPCs) under shear stress on the biological function such as proliferation, adhesion, migration, apoptosis and expression of α-smooth muscle actin (α-SMA), collagen-I and collagen-Ⅲ of hepatic stellate cells (HSCs). METHODS: HSCs and EPCs were inoculated into the upper and lower layers of the co-culture chamber respectively and co-incubated for 24 hours. Then, 12 dyne/cm2 shear stress was applied to EPCs cells for another 24 hours. After that, proliferation, adhesion, migration and apoptosis of HSCs were detected by cell counting kit-8 (CCK-8) kit, cell adherent assay, Boyden cell migration assay and flow cytometry respectively. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression of alpha -SMA, collagen I and collagen-Ⅲ in HSCs. RESULTS: Under shear stress, EPCs ecological niche could obviously inhibit the proliferation, adhesion and migration of HSCs, promote the apoptosis of HSCs, and down-regulate the mRNA and protein expression of collagen-I, collagen-Ⅲ in HSC cells. CONCLUSIONS: Under shear stress, EPCs ecological niche could inhibit the fibrosis development of HSCs to a certain extent.

[The role of mannose binding lectin in the pathogenesis of systemic lupus erythematosus].

OBJECTIVE: To detect the serum level of mannose binding lectin (MBL) and its genovariation in systemic lupus erythematosus (SLE) patients and to investigate the role of MBL in the pathogenesis of SLE. METHODS: ELISA was used to measure the serum MBL level of 40 SLE patients and 30 healthy blood donors. Tm genotyping method was used for the first timer in China. Primers and specific fluorophore-labelled hybridization probes for the exon 1 and promoter regions of MBL gene were designed based on the haplotype MBL2(*) LXPA (GenBank X15422). The genotyping of MBL in these two groups were performed using real-time PCR through Light Cycler Instrument. RESULTS: (1) The serum MBL of the SLE patients was 107.2 microg/L, significantly lower than that of the healthy blood donors (290.2 microg/L, P = 0.0002). (2) MBL mutation in exon 1 region was mainly at codon 54, with a mutation rate of 37.1% in the SLE group, significantly higher than that of the control group (13.3%, p = 0.049). (3) Polymorphisms of H/L in MBL gene were present in both SLE patients and controls, and there was no difference in the L allele frequency between the two groups. (4) The serum MBL level of the SLE patients with MBL mutation in codon 54 was 49.8 microg/L, significantly lower than that of the SLE patients without MBL mutation in codon 54 (141.7 microg/L, P = 0.000 27). The SLE disease activity index (SLEDAI) of the SLE patients with MBL mutation in codon 54 was 7.44, significantly lower than that of the SLE patients without MBL mutation in codon 54 (12.87, P = 0.0029). A negative correlation was observed between SLEDAI score and serum MBL (r = -0.48). CONCLUSION: Mutation occurring in MBL exon 1 region at codon 54 may be a predisposing factor of the pathogenesis of SLE. Serum MBL may be a potential biomarker of disease activity in SLE patients.