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

Abstract

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.

Alphavirus RNA synthesis and non-structural protein functions

“…The TATase activity observed with the N-terminal truncated nsP4 was independent of other viral factors; however, a predilection towards viral RNA substrates was observed. Whether or not this is the genuine mechanism for viral polyadenylation is still unknown; evidence indicating the presence of a 59 poly(U) tract on the minus-strand RNA and the ability of truncated alphavirus genomic RNAs to be polyadenylated during infection highlight the complexity of viral polyadenylation (Hill et al, 1997;Raju et al, 1999;Sawicki & Gomatos, 1976). Purification of full-length nsP4 has been accomplished using an N-terminal SUMO tag method.…”

“…However, it is also possible that both a templated and non-templated mechanism of polyadenylation and terminal addition are at play during an alphavirus infection, allowing repair and restoration of infectivity of damaged or defective viral genomes. Further work by Raju and colleagues has found that, despite the essential nature of the 39 UTR, genomes lacking significant portions of this element are capable of producing progeny by a novel repair mechanism (George & Raju, 2000;James et al, 2007;Raju et al, 1999). Synthetic genomes with a set of deletions to the 39 CSE, UTR and/or poly(A) tail were introduced into cells, and viable progeny were recovered from many of these.…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…Various deletions and insertions in the 3= UTR as well as complete deletion of the poly(A) tail may not kill alphaviruses (457)(458)(459), flaviviruses (460)(461)(462), coronaviruses (463)(464)(465), and various plant viruses (466)(467)(468)(469). The repair of the damaged genomes may again involve different mechanisms, including RNA recombination, the use of the viral RdRP-or host-dependent polyadenylation activities, and perhaps some others.…”

Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD)

“…Alphaviruses possess a highly conserved 3' sequence element (3' CSE; approximately 19 nt long) that immediately precedes the poly(A) tail [2]. Both the poly(A) tail and the 3'CSE are required for virus replication and, more specifically, for efficient minus-strand RNA synthesis [13][14][15][16][17]. The terminal 19 nt conserved sequence was identical in all GETV isolates, including the M1 isolate that was previously reported to have lost this conserved sequence [18,19].…”