In vivo addition of poly(A) tail and AU-rich sequences to the 3 ' terminusof the sindbis virus RNA genome: A novel 3 '-end repair pathway

Alphaviruses are mosquito transmitted RNA viruses that cause important dise ases 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 n egative-sense RNA and genome replication in vivo. We have recently demonstr ated that the SIN genome lacking the poly(A) tail was infectious and that d e novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J, Hu, a nd 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 cytop lasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations i n the 3'NTR and tested their ability to generate infectious virus and under go 3' polyadengation in vivo, Engineered SIN genomes with terminal deletion s within the 19-nt 3'CSE were infectious and regained their poly(A) tail. A lso, a SIN genome carrying the poly(A) tail but lacking a part or the entir e 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated f rom 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 in fected host cell. Most importantly, these results indicate the ability of a lphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis a nd genome replication in vivo, The possible roles of cytoplasmic polyadenyl ation machinery, terminal transferase-like enzymes, and the viral polymeras e in the terminal repair processes are discussed.