基因型1型戊型肝炎病毒(HEV-1),与其他基因型的HEV不同,具有称为ORF4的独特小开放阅读框,其功能尚不清楚。ORF4以框架外的方式位于ORF1的中间,ORF1根据菌株编码推定的90至158个氨基酸。探讨ORF4在HEV-1复制和感染中的作用,我们克隆了T7RNA聚合酶启动子下游的野生型HEV-1的完整基因组,制备了以下ORF4突变构建体:第一个构建体具有TTG而不是起始密码子ATG(A2836T),在ORF4中引入M→L突变,在ORF1中引入D→V突变。第二个构建体具有ACG而不是ATG密码子(T2837C),在ORF4中引入M→T突变。第三个构建体具有ACG而不是第二个框内ATG密码子(T2885C),在ORF4中引入M→T突变。第四个构建体含有两个突变(T2837C和T2885C),伴随ORF4中的两个M→T突变。对于后三种结构,ORF1中引入的伴随突变均为同义变化.通过体外转录产生加帽的整个基因组RNA,并用于转染PLC/PRF/5细胞。ORF1中含有同义突变的三个mRNA,即T2837CRNA,T2885CRNA,和T2837C/T2885CRNA,在PLC/PRF/5细胞中正常复制,并产生与野生型HEV-1一样成功感染蒙古沙鼠的感染性病毒。相比之下,突变的RNA,即,A2836TRNA,伴随着ORF1中的氨基酸变化(D937V)在转染后产生的感染性病毒,但是它们的复制速度比野生型HEV-1慢,并且未能感染蒙古沙鼠。通过使用高滴度抗HEV-1IgG抗体的Western印迹分析,在野生型HEV-1-以及突变病毒感染的PLC/PRF/5细胞中未检测到源自ORF4的推定病毒蛋白。这些结果表明,ORF4缺陷的HEV-1s具有在培养细胞中复制的能力,这些有缺陷的病毒有能力感染蒙古沙鼠,除非重叠的ORF1伴有非同义突变,确认ORF4在HEV-1的复制和感染中不是必需的。
Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.