| Summary: | 碩士 === 國立中興大學 === 獸醫病理生物學研究所 === 102 === Coronavirus is an enveloped, positive-sense, single-stranded RNA virus, with the largest genome size of 26.4 kilobase (kb) to 31.7 kb among the known RNA viruses. The AAUAAA motif located in the 3’UTR of eukaryotic mRNA is an important signal for polyadenylation. Since the genome structure of coronavirus is similar to that of eukaryotic mRNA and the AAUAAA motif or its variation AGUAAA is also similarly found in the 3’UTR of betacoronaviruses, we hypothesized that the AGUAAA sequence may play an important role in coronavirus polyadenylation. For this, we employed coronavirus defective interfering RNA (DI RNA) to construct a series of AGUAAA mutants with different length of poly(A) tail, and the mutated DI RNA was then transfected into Human rectal tumor cell (HRT-18 cell). The length of poly(A) tail was determined by head-to-tail ligation followed by RT-PCR and sequencing. The results were as follows: (1) the AGUAAA sequence was involved in coronaviral polyadenylation. (2) Coronavirus with the length of 15 to 20 nucleotides (nts) poly(A) tail still maintained the polyadenylated ability even though the AGUAAA motif was mutated. (3) The synergy of AGUAAA motif and at least 15 to 20-nts of poly(A) tail has the best efficiency for the polyadenylation of coronavirus during infection. Therefore, based on the results we propose that the coronavirus polyadenylation occurs during positive-strand RNA synthesis and involves the AGUAAA motif and related proteins.
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