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Journal of Virology, March 1999, p. 2410-2419, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3' Terminus of the Sindbis Virus RNA Genome: a Novel 3'-End Repair Pathway

Ramaswamy Raju,* Mustapha Hajjou, Kristie R. Hill, Vandana Botta, and Sisir Botta

Department of Microbiology, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208

Received 10 September 1998/Accepted 7 December 1998

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.

* Corresponding author. Mailing address: Department of Microbiology, Meharry Medical College, School of Medicine, 1005 D.B. Todd Blvd., Nashville, TN 37208. Phone: (615) 327 6687. Fax: (615) 327 6602. E-mail: ramasa25{at}ccvax.mmc.edu.

Journal of Virology, March 1999, p. 2410-2419, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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