Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication.

IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.

丙型肝炎病毒（HCV）感染已被证明可调节患者活检标本和培养细胞中的microRNA 130a（miR-130a）。我们试图鉴定miR-130a靶基因并探索miR-130a调节HCV和乙型肝炎病毒（HBV）复制的机制。我们使用了包括miRanda，TargetScan，PITA和RNAhybrid在内的生物信息学软件来预测潜在的miR-130a靶基因。通过使用小干扰RNA（siRNA）或成簇规则间隔的短回文重复序列（CRISPR）/ Cas9指导RNA（gRNA），miR-130a及其靶基因被过表达或敲低。选定的基因mRNA及其蛋白以及HCV在OR6细胞，HCV JFH1感染的Huh7.5.1细胞和HCV JFH1感染的原代人肝细胞（PHH）中的复制以及在HepAD38细胞，HBV感染的NTCP-Huh7.5.1中的HBV复制。细胞，分别通过定量逆转录PCR（qRT-PCR）和Western印迹检测HBV和HBV感染的PHH。我们选择了116个预测的靶基因，它们的表达与病毒的发病机制或免疫相关，用于qPCR验证。其中，证实肝和红细胞（PKLR）中编码丙酮酸激酶的基因受miR-130a过表达的调节。miR-130a过表达（通过模拟）降低了PKLR mRNA和蛋白水平。miR-130a抑制剂和gRNA增加PKLR表达，HCV复制和HBV复制，而miR-130a gRNA和PKLR过表达增加HCV和HBV复制。补充丙酮酸增加了HCV和HBV复制，并通过miR-130a模拟物和PKLR敲低挽救了对HCV和HBV复制的抑制作用。我们得出的结论是，miR-130a通过靶向PKLR和随后产生丙酮酸来调节HCV和HBV复制。我们的数据提供了对miR-130a调控的关键代谢酶途径的新颖见解，包括涉及PKLR和丙酮酸的步骤，这些步骤被HCV和HBV复制所破坏。

重要我们确定miR-130a调节靶基因PKLR及其随后对丙酮酸产生的影响。丙酮酸是几种代谢途径的关键中间体，我们确定丙酮酸在调节HCV和HBV复制中起关键作用。这种先前无法识别的，miRNA调节的抗病毒机制对宿主定向策略的开发具有影响，该策略可中断病毒生命周期并防止建立针对HCV，HBV和潜在其他病毒感染的持续感染。