是所有丙型肝炎病毒（HCV）基因型和亚基因型中最保守的区域，HCV基因组的5'非翻译区（5'UTR）表明它作为基于RNA干扰的抗mRNA治疗策略的潜在靶标的重要性。第一个基于小干扰RNA（siRNA）的遗传性转甲状腺素蛋白介导的淀粉样变性治疗的临床应用的出现和批准，提高了对这种方法对抗高度易感病毒如HCV的希望。我们调查了来自沙特人群的HCV亚型4a分离株的5'UTR内的内部核糖体进入位点（IRES）结构的茎环（SLs）II和III核苷酸基序的共有siRNA的抗病毒潜力。siRNA对病毒复制和全长HCV基因组翻译的抑制作用在一个主管，持久性,和可再生的Huh-7细胞培养系统维持一个月。通过在Huh-7细胞中靶向IRES的SL-III核苷酸基序的siRNA证明了HCV-IRES克隆的RNA转录物水平的最大抑制以及病毒复制和全长病毒基因组翻译的沉默。siRNAUsi-169在转染后24小时降低了HCV-IRES克隆的5'UTRRNA转录本水平，在转染后24小时降低了75％（P<0.001），在48小时处理时降低了80％（P<0.001）Huh-7细胞。在24小时和48小时的时间跨度，与构建的载体（即pCR3.1/GFP/5'UTR）和siRNAUsi-169共转染的Huh-7细胞中，5'UTR标记的GFP蛋白表达从70％显着降低至80％。在持续的Huh-7细胞培养系统中，胞浆内siRNAUsi-169活性也显着降解了病毒复制被90％以上（P<0.001）和HCV核心（C）和高变包膜糖蛋白（E1和E2）表达。研究结果揭示，靶向HCV亚型4a沙特分离株的5'UTR-IRES的siRNA在持续的体外Huh-7组织培养系统中显示出HCV复制的有效沉默和病毒翻译的阻断。此外，我们还阐明，siRNA沉默病毒mRNA不仅抑制病毒复制，而且阻断病毒翻译。结果表明，siRNA有效的抗病毒活性应被认为是一种有效的基于抗mRNA的治疗策略，用于针对沙特阿拉伯研究较少和难以治疗的HCV亚型4a分离株的进一步体内研究。
Being the most conserved region of all hepatitis C virus (HCV) genotypes and sub-genotypes, the 5\' untranslated region (5\' UTR) of HCV genome signifies it\'s importance as a potential target for anti-mRNA based treatment strategies like RNA interference. The advent and approval of first small interference RNA (siRNA) -based treatment of hereditary transthyretin-mediated amyloidosis for clinical use has raised the hopes to test this approach against highly susceptible viruses like HCV. We investigated the antiviral potential of
consensus siRNAs targeted to stem-loops (SLs) II and III nucleotide motifs of internal ribosome entry site (IRES) structure within 5\' UTR of HCV sub-genotype 4a isolates from the Saudi population. siRNA inhibitory effects on viral replication and translation of full-length HCV genome were determined in a competent, persistent, and reproducible Huh-7 cell culture system maintained for one month. Maximal inhibition of RNA transcript levels of HCV-IRES clones and silencing of viral replication and translation of full-length virus genome was demonstrated by siRNAs targeted to SL-III nucleotide motifs of IRES in Huh-7 cells. siRNA Usi-169 decreased 5\' UTR RNA transcript levels of HCV-IRES clones up to 75% (P < 0.001) at 24 h post-transfection and 80% (P < 0.001) at 48 h treatment in Huh-7 cells. 5\' UTR-tagged GFP protein expression was significantly decreased from 70 to 80% in Huh-7 cells co-transfected with constructed vectors (i.e. pCR3.1/GFP/5\' UTR) and siRNA Usi-169 at 24 h and 48 h time-span. Viral replication was inhibited by more than 90% (P < 0.001) and HCV core (C) and hypervariable envelope glycoproteins (E1 and E2) expression was also significantly degraded by intracytoplasmic siRNA Usi-169 activity in persistent Huh-7 cell culture system. The findings unveil that siRNAs targeted to 5\' UTR-IRES of HCV sub-genotype 4a Saudi isolates show potent silencing of HCV replication and blocking of viral translation in a persistent in-vitro Huh-7 tissue culture system. Furthermore, we also elucidated that siRNA silencing of viral mRNA not only inhibits viral replication but also blocks viral translation. The results suggest that siRNA potent antiviral activity should be considered as an effective anti-mRNA based treatment strategies for further in-vivo investigations against less studied and harder-to-treat HCV sub-genotype 4a isolates in Saudi Arabia.
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