血红素加氧酶1抑制猪繁殖与呼吸综合征病毒复制的分子机制

Porcine reproductive and respiratory syndrome(PRRS) is one of the most economically important swine viral infectious diseases affecting swine industry worldwide. However, current antiviral strategy could not effectively prevent and control this disease. Our recent studies have demonstrated that induction or over-expression of heme oxygenase 1 (HO-1) inhibited PRRS virus (PRRSV) replication in PRRSV permissive cells, Marc-145, PK-15CD163, and primary alveolar macrophages (PAM). Our hypothesis is that HO-1 also inhibit PRRSV replication in pig. What are the molecular mechanisms of HO-1 in the inhibition of PRRSV replication ? To test this hypothesis, firstly, we propose to test the ability of HO-1 to inhibit PRRSV replication in pig. Then we determine the specific transcription factors regulating HO-1 gene expression and PRRSV replication, and define PRRSV proteins interacting with HO-1 and their role during PRRSV infection by employing over-expression, RNA interference (RNAi), Co-Immunoprecipitation (Co-IP), Bimolecular Fluorescence Complementation (BiFC), Confocal colocalization, and other techniques. The proposed studies are expected to provide additional information for better understanting the molecular mechanisms of HO-1 in inhibiting PRRSV replication from the aspects both transcriptional regulation and virus-host interaction. The results generated from this project will pave the way for elucidating the molecular machnisms of PRRSV pathogenesis and developing HO-1 based novel approach for the prevention and control of PRRS.

猪繁殖与呼吸综合征（PRRS）是危害全球养猪业的重大病毒性疫病，但目前对该病的预防和控制尚未取得突破性进展。我们发现血红素加氧酶1（HO-1）显著抑制PRRSV在其易感细胞Marc-145和PK-15CD163及宿主靶细胞PAM中复制。因此推测HO-1也可能抑制该病毒在猪体内复制。HO-1抑制PRRSV复制的分子机制是什么？为了回答这一重要科学问题，本研究首先在猪体内验证HO-1抑制PRRSV复制；然后运用高表达、RNAi、Co-IP、BiFC、FACS和激光共聚焦等方法，通过解析调控HO-1表达进而抑制PRRSV复制的上游关键转录因子、鉴定与HO-1互作的下游PRRSV蛋白并明确二者互作对病毒复制的影响，从宿主基因的转录调控及病毒蛋白与宿主蛋白相互作用的角度深入研究HO-1抑制PRRSV复制的分子机制。为进一步阐明PRRSV的分子致病机制并确立HO-1作为PRRS防治新靶点提供科学依据。

猪繁殖与呼吸综合征（PRRS）是危害全球养猪业的重大病毒性疫病。由于对PRRS病毒（PRRSV）感染与复制的分子调控机制缺乏深入认识，目前对该病的预防和控制尚未取得突破性进展。我们前期研究发现宿主因子血红素加氧酶1（HO-1）能够抑制PRRSV在细胞中复制。但HO-1抑制PRRSV复制的分子机制尚不清楚。该项目首先从猪体内外研究证实了HO-1确实能够抑制不同毒力PRRSV毒株的感染与复制，而且HO-1还能够抑制BVDV复制，表明HO-1可能具有广谱抗病毒功能；然后克隆获得了猪HO-1基因启动子区，鉴定到了核心启动子区域，预测筛选了核心启动子区的转录因子结合位点，证明转录因子Nrf2能够特异性结合到猪HO-1基因的启动子区增强HO-1表达进而抑制PRRSV复制；从转录后microRNA角度证明miR-24-3p和miR-22直接靶向HO-1的3’UTR，同时通过mRNA降解和翻译抑制两种方式抑制HO-1的表达进而实现自身的感染与复制；HO-1不是通过与PRRSV的蛋白质直接相互作用发挥抗病毒感染功能，主要通过其下游代谢产物胆绿素和一氧化碳而非铁离子发挥抗病毒功能；胆绿素发挥抗PRRSV感染作用主要由胆绿素的二级代谢产物胆红素介导并通过一氧化氮依赖的cGMP/PKG信号通路实现；一氧化碳发挥抗PRRSV感染作用主要通过激活细胞的cGMP/PKG信号通路并负调控PRRSV复制所必需的NF-κB信号通路实现。研究成果对深入解析HO-1调控PRRSV感染与复制的分子机制具有重要科学意义，也为确立HO-1作为PRRS防治的新靶点提供了科学依据。