Cleavage of coronavirus RNA is associated with cellular endoribonuclease RNase L

• Main Authors: Hsuan-Yung Lin, 林瑄詠
• Other Authors: Hung-Yi Wu
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/xmz73c

碩士 === 國立中興大學 === 獸醫病理生物學研究所 === 106 === Among the cellular endoribonucleases, RNase L is a well-studied endoribonuclease associated with antiviral defense induced by innate immunity. RNase L cleaves viral and host RNA including 28S and 18S rRNA predominantly after single-stranded UA and UU dinucleotides. In coronaviruses, the cleavage of coronaviral RNA and the features for the cleavage preference by RNase L has not been previously probed. In an attempt to test whether coronavirus defective interfering (DI) RNA with transcription regulating sequence (TRS) is able to synthesize subgenomic DI RNA 12.7 (sgmRNA 12.7), it was unexpectedly found by Northern blot assay that, in addition to predicted sgmRNA 12.7, an RNA fragment designed ST RNA with a size less than sgmRNA 12.7 was also found. Subsequent study demonstrated that the cleaved site for the ST RNA is located downstream of 12.7 sgmRNA TRS, in the loop region of stem-loop II and after UU dinucleotides. The cleaved DI RNA 12.7 fragment ST RNA was identified with Northern blot assay in both uninfected and bovine coronavirus (BCoV)-infected HRT18-cells, indicating that the cellular factors are responsible for the cleavage. The similar cleavage was also found in HEK-293T and A549 cells. To characterize the features of the cleavage, mutagenesis followed by Northern blot assay was performed. It was found that the cellular factor preferentially cleaved after UU or UA dinucleotides, and the sequences upstream and downstream of UU dinucleotides has influence on the efficiency of the cleavage, consisting with the general criteria of cleavage by cellular RNase L. Since the cleavage of 28S and 18S rRNA indicates the activation of RNase L, the cleavage of rRNA as well as DI RNA 12.7 found in A549 cells suggested that the cleavage of DI RNA 12.7 is correlated to RNase L. In conclusion, we for the first time demonstrated that the cleavage of coronavirus genome is correlated to RNase L. Accordingly, since similar cleavage was also found in cells (HRT-18 and HEK-293T cells) from which RNase L are not inducible, it is proposed that these cells may have basal levels of activated RNase L, which is independent of innate immunity and may serve as a non-specific role for the fast cleavage of foreign single-stranded RNA.