R. Raju et al., 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, J VIROLOGY, 73(3), 1999, pp. 2410-2419
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.