School of Life Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea.
J Microbiol Biotechnol. 2019 Nov 28;29(11):1790-1798. doi: 10.4014/jmb.1906.06054.
Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'- end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28°C but weak at 30°C. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.
蜂房病毒（FHV）是一种昆虫 RNA 病毒，具有二分体基因组。只要 FHV RNA 的 3' 末端具有 TYMV 序列，FHV RNA1 就可以被芜菁黄花叶病毒（TYMV）包装。包装的 FHV RNA1 在 5' 末端有四个额外的核苷酸。我们研究了重组 FHV RNA1 是否可以在哺乳动物细胞中复制。为了解决这个问题，我们制备了模拟重组 FHV RNA1 的体外转录 FHV RNA，并将其导入仓鼠肾细胞（BHK）。结果表明，重组 FHV RNA1 能够复制。插入到 FHV RNA1 的 B2 基因中的 eGFP 基因也成功表达。我们还观察到，在 28°C 时，eGFP 在蛋白质水平上的表达很强，但在 30°C 时较弱。序列分析表明，RNA1 和 RNA3 复制产物的 3' 末端与真实 FHV RNA 的 3' 末端相同。这表明 FHV 复制酶正确识别了内部的复制信号。相比之下，重组 FHV RNA1 的 5' 末端经常发生缺失，表明（+）链合成的随机起始。
