Molecular determinants of mouse adaptation of rat hepacivirus

Lack of immunocompetent animal models for hepatitis C virus (HCV) impedes vaccine development and studies of immune responses. Norway rat hepacivirus (NrHV) infection in rats shares HCV-defining characteristics, including hepatotropism, chronicity, immune responses, and aspects of pathology. To exploit genetic variants and research tools, we previously adapted NrHV to prolonged infection in laboratory mice. Through intrahepatic RNA inoculation of molecular clones of identified variants, we here characterized four mutations in the envelope proteins responsible for mouse adaptation, including one disrupting a glycosylation site. These mutations led to high titer viremia, similar to that observed in rats. In 4-week-old mice, infection was cleared after around 5 weeks compared to 2-3 weeks for non-adapted virus. In contrast, the mutations led to persistent but attenuated infection in rats, and they partially reverted accompanied by an increase in viremia. Attenuated infection in rat but not mouse hepatoma cells demonstrated that the characterized mutations were indeed mouse adaptive rather than generally enhancing fitness in both cell lines, and that species determinants and not immune interactions were responsible for attenuation in rats. Unlike persistent NrHV infection in rats, acute resolving infection in mice was not associated with development of neutralizing antibodies. Finally, infection of scavenger receptor B-I (SR-BI) knockout mice suggested that adaptation to mouse SR-BI was not a primary function of the identified mutations. Rather, the virus may have adapted to lower dependency on SR-BI, thereby potentially surpassing species-specific differences. In conclusion, we identified specific determinants of NrHV mouse adaptation, suggesting species-specific interactions during entry.丙型肝炎病毒（hepatitis C virus, HCV）缺乏免疫健全动物模型，这一短板阻碍了疫苗研发与免疫应答相关研究。挪威大鼠肝炎病毒（Norway rat hepacivirus, NrHV）感染大鼠后，可呈现HCV的典型特征，包括嗜肝性、慢性化进程、免疫应答及部分病理表现。为利用基因变异体与现有研究工具，本团队此前已将NrHV适配为可在实验小鼠中建立持续感染的毒株。本研究通过对已鉴定变异体的分子克隆进行肝内RNA接种，鉴定出包膜蛋白上与小鼠适配相关的4处突变，其中1处突变可破坏糖基化位点。这些突变可使小鼠体内出现高滴度病毒血症，其水平与大鼠感染NrHV时的病毒血症水平相当。与非适配毒株感染后2~3周即被清除相比，4周龄小鼠感染适配毒株后，感染状态约在5周左右才被清除。与之相反，这些突变会使大鼠出现持续但减毒的感染，且随着病毒血症水平升高，部分突变发生了回复突变。大鼠肝癌细胞（而非小鼠肝癌细胞）可出现减毒感染，这表明本研究鉴定的突变确实为小鼠适配性突变，而非同时提升两种细胞系中的病毒复制能力；同时证实大鼠体内的感染减毒由物种特异性决定因素介导，而非免疫互作导致。与大鼠体内持续的NrHV感染不同，小鼠体内的急性自限性感染并未伴随中和抗体的产生。最后，对清道夫受体B-I（scavenger receptor B-I, SR-BI）敲除小鼠的感染实验表明，适配小鼠SR-BI并非上述鉴定突变的主要功能。相反，病毒可能通过这些突变适配为对SR-BI的依赖性降低，从而潜在地突破了物种特异性屏障。综上，本研究鉴定出NrHV小鼠适配的特异性决定因素，提示病毒入侵过程中存在物种特异性的互作机制。