Raju R, Hajjou M, Hill K R, Botta V, Botta S
Department of Microbiology, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208, USA.
J Virol. 1999 Mar;73(3):2410-9. doi: 10.1128/JVI.73.3.2410-2419.1999.
Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5' end and polyadenylated at its 3' end. The 3' nontranslated region (3'NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3'CSE) and a poly(A) tail. This 3'CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693-2704, 1997). Here, we demonstrate that the 3'-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations in the 3'NTR and tested their ability to generate infectious virus and undergo 3' polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3'CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3'NTR. These results indicate the ability of alphavirus RNAs to undergo 3' repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3' end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.
甲病毒是由蚊子传播的RNA病毒，可在人类和牲畜中引发重要疾病。辛德毕斯病毒（SIN）是甲病毒属的模式种，携带一个11.7 kb的正义RNA基因组，其5'端有帽状结构，3'端有多聚腺苷酸化。SIN基因组的3'非翻译区（3'NTR）有许多富含AU的基序，包括一个19个核苷酸（nt）的保守元件（3'CSE）和一个多聚（A）尾。据信，这个3'CSE和相邻的多聚（A）尾在体内调节负义RNA的合成和基因组复制。我们最近证明，缺乏多聚（A）尾的SIN基因组具有感染性，并且在体内可以发生从头多聚腺苷酸化（K. R. Hill、M. Hajjou、J. Hu和R. Raju，《病毒学杂志》71:2693 - 2704，1997）。在此，我们证明SIN基因组的3'末端29 nt区域携带一个可能用于细胞质多聚腺苷酸化的信号。为了进一步研究3'NTR内的多聚腺苷酸化信号，我们构建了一系列在3'NTR有突变的突变基因组，并测试它们在体内产生感染性病毒和进行3'多聚腺苷酸化的能力。在19 nt的3'CSE内有末端缺失的工程化SIN基因组具有感染性，并重新获得了它们的多聚（A）尾。此外，一个携带多聚（A）尾但缺少部分或全部19 nt 3'CSE的SIN基因组也具有感染性。对由这些工程化SIN基因组产生的病毒进行的序列分析表明，在多聚（A）尾附近添加了各种富含AU的序列基序。向突变的SIN基因组中添加富含AU的基序似乎需要3'NTR的很大一部分存在。这些结果表明甲病毒RNA具有进行3'修复的能力，以及在受感染宿主细胞中存在一条向其3'端添加富含AU序列和多聚（A）尾的途径。最重要的是，这些结果表明甲病毒复制机制能够利用大量由多聚（A）基序邻接的富含AU的RNA序列作为体内负义RNA合成和基因组复制的启动子。文中讨论了细胞质多聚腺苷酸化机制、末端转移酶样酶和病毒聚合酶在末端修复过程中的可能作用。
