Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly.

IMPORTANCE HCV DVGs have been identified in vivo and in vitro, but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We identified a highly conserved secondary structure (SL750) within the core-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed previously unexpected novel cis elements critical for HCV replication and morphogenesis.

丙型肝炎病毒（HCV）的病毒基因组（DVGs）存在缺陷，但其生物学意义尚未得到充分研究。在这里，我们分析了在患者血清中循环的HCV DVG，这些病毒在结构蛋白编码区中具有缺失。在41例HCV临床分离株中，约30％的DVG来源于同一患者的全长基因组。DVG，病毒血症和丙氨酸氨基转移酶（ALT）水平之间没有相关性。DVG的测序分析显示存在缺失热点，在E1的上游位点以及在E2和NS2的下游位点。有趣的是，NSG2核心蛋白和C末端蛋白酶结构域的编码序列在DVG中始终是完整的，尽管事实上这两种蛋白对于HCV基因组复制都是可有可无的。机理研究表明，NS2的跨膜区段3（TMS3）位于其蛋白酶结构域的上游，是NS2-NS3裂解和DVG复制所必需的。此外，我们在核心域2编码区域内发现了高度保守的二级结构（SL750），这对于HCV基因组包装至关重要。总而言之，我们对血清来源的HCV DVG的分析揭示了新型病毒在病毒复制和组装中起重要作用的顺式元件。

重要性HCV DVGs已经确定在体内和体外，但它们的生物合成和生理意义仍然是难以捉摸的。另外，尚未在HCV RNA基因组上鉴定出常规的包装信号，并且HCV基因组被衣壳化成感染性颗粒的特异性基础的机制尚待发现。在这里，我们确定了新的病毒顺式通过确定定义血清来源的HCV DVG边界的遗传限制，对HCV生命周期至关重要的元素。我们发现NS2的跨膜片段3，位于其蛋白酶结构域的上游，是NS2-NS3的裂解和DVGs复制所必需的。我们在核心编码区内鉴定了高度保守的二级结构（SL750），这对于HCV基因组包装至关重要。总而言之，我们对血清来源的HCV DVG的分析揭示了以前未曾预料到的对HCV复制和形态发生至关重要的新型顺式元件。