Different Gene Networks Are Disturbed by Zika Virus Infection in A Mouse Microcephaly Model

The association of Zika virus (ZIKV) infection with microcephaly has raised alarm worldwide. Their causal link has been confirmed in different animal models infected by ZIKV. However, the molecular mechanisms underlying ZIKV pathogenesis are far from clear. Hence, we performed global gene expression analysis of ZIKV-infected mouse brains to unveil the biological and molecular networks underpinning microcephaly. We found significant dysregulation of the sub-networks associated with brain development, immune response, cell death, microglial cell activation, and autophagy amongst others. We provided detailed analysis of the related complicated gene networks and the links between them. Additionally, we analyzed the signaling pathways that were likely to be involved. This report provides systemic insights into not only the pathogenesis, but also a path to the development of prophylactic and therapeutic strategies against ZIKV infection.     

Ku70作为RNA解旋酶调节miR-124的加工成熟及神经细胞的分化

目的 Ku70蛋白主要通过其DNA结合特性参与双链DNA断裂（DSB）的非同源端连接（NHEJ）修复,有报道称其具有RNA结合功能,本文探索Ku70是否具有RNA解旋酶活性并影响miRNA加工成熟。方法 利用RNA免疫共沉淀（RIP）测序结合生物信息学分析Ku蛋白结合的RNA;蛋白质印迹法（Western blot,WB）结合定量反转录PCR（qRT-PCR）检测Ku蛋白与miRNAs的表达关系;生物膜干涉技术（BLI）实验分析Ku蛋白与RNA的结合能力;电泳迁移率变动分析（EMSA）实验确定Ku70及Ku80的RNA解旋酶活性;形态学检测结合WB分析Ku70调节miR-124引起的神经细胞功能变化;免疫荧光结合形态学分析寨卡病毒（ZIKV）感染后Ku70及miR-124的变化与神经元分化关联。结果 研究发现,Ku70蛋白具有RNA解旋酶活性,并通过其RNA解旋酶活性影响miRNA加工成熟。Ku70缺失引起许多miRNAs上调,其中包括神经细胞特异的miR-124。在人神经前体细胞（hNPCs）和人神经母细胞瘤细胞（SH-SY5Y）中敲低Ku70可促进 miR-124的成熟,从而导致上述细胞向神经元分化。本文进一步发现,ZIKV感染影响了Ku70及miR-124的表达,导致细胞形态的分化。结论 本研究揭示了Ku70的一种新功能,即Ku70有可能参与miRNA的成熟调控和神经细胞的分化,并且可能是ZIKV病毒致小头症的原因之一。     

Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

Kaposi’s sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi’s sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.     

Design,synthesis, and evaluation of novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile compounds as Zika inhibitors

The prevalence of Zika virus (ZIKV) has become widespread in recent years. ZIKV infection is associated with severe congenital CNS malformations in both newborns and adults. However, neither vaccines nor therapeutics are available to control ZIKV infection until now. We started by hit screening our in-house small molecule library, then designed, synthesized, and evaluated a new class of 1, 4-bibenzylsubstituted piperazine derivatives for their cytopathic effect (CPE) protection effect in a ZIKV-infected Vero E6 cellular assay. A preliminary structure–activity relationship study identified five novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile analogs with obvious CPE reduction effects against ZIKV at micromolar concentrations. Moreover, compound 3p exerted a significant antiviral effect on both Zika RNA replication and virus protein expression in a dose-dependent manner at low micromolar concentrations. This study demonstrated the potential of a novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile scaffold for the development of anti-ZIKV candidates.     

猪伪狂犬病毒Fa株侵染对PK-15细胞microRNAs表达谱的影响

【目的】分析猪伪狂犬病毒Fa株(PRV-Fa)侵染对猪肾传代细胞PK-15 microRNAs(miRNAs)表达谱的影响。【方法】利用Illumina高通量测序技术,鉴定感染和非感染PRV-Fa的PK-15细胞的miRNAs;筛选并利用实时荧光定量RT-PCR(RT-q PCR)验证差异表达miRNAs;对差异miRNAs进行靶基因预测和Gene ontology(GO)分析。【结果】在感染和未感染PK-15细胞中分别检测到384个和405个miRNAs,其中感染PRV-Fa后差异表达的miRNAs共127个(60个上调,67个下调)。荧光定量结果显示差异miRNAs的表达趋势与高通量测序结果一致。GO分析显示,miRNAs广泛参与信号传导、细胞代谢、免疫反应、基因表达等生物学进程,其中miR-10b、miR-16、miR-18a、miR-19b、miR-20a、miR-145-5p、miR-146a、miR-181a、miR-499-5p等miRNAs与免疫相关。在靶基因调控网络图中,ssc-miR-30a-5p与ssc-miR-30d处于关键位置。研究鉴定出5个新的病毒编码miRNAs,其中PRV-miR-LLT2与PRV-miR-LLT4靶向PRV早期蛋白基因EPO。【结论】伪狂犬病毒Fa株感染对PK-15细胞编码miRNAs有显著影响。