3'-End Repair

We have recently discovered three different repair activities for the TCV RdRp. Many positive-stranded RNA viruses contain short, single-stranded 3'-ends that are vulnerable to degradation by host cellular RNases. Therefore, the existence of 3'-end repair mechanisms (analogous to cellular telomerases) must be required and/or advantageous for RNA viruses. TCV genomic and subviral RNAs terminate with the single-stranded motif CCUGCCC-3'. Deletions of 3 to 5 bases of the satellite RNA satC are repaired in vitro and in vivo by non-template addition of bases by the RdRp, with a preference for multiple consecutive bases. The repaired sequences can differ substantially from the wild-type sequence.   Deletion of 3 bases connected to 8 non-template bases is repaired by homologous recombination with the similar 3' end region of the TCV genomic RNA.  Deletion of 6 or 7 bases are repaired to the wild-type sequence in vivo and in vitro. This repair mechanism apparently involves the production of 4 to 8 nt oligoribonucleotides by abortive synthesis by the RdRp using the 3' end of TCV genomic RNA as template. Based on in vitro results, specific oligoribonucleotides are able to prime synthesis of wild-type minus-strand satC in a reaction that does not require base-pairing of the oligoribonucleotide to the truncated, positive-strand RNA template but does require the hairpin that is necessary for normal satC minus-strand synthesis. SatD, with deletions of 6 to 11 bases is also repaired in vivo. Repaired satD have additional deletions to the -14 position joined to internal TCV genomic RNA (or other sequence) followed by replacement of the terminal CUGCCC-3' motif. Selection of internal TCV sequences is not random and is likely facilitated by base-pairing between internal regions of TCV genomic RNA and the oligoribonucleotides generated by abortive cycling from the 3' end of the TCV genome. Continuation of this project involves determining the sequences/structures required for abortive oligonucleotide synthesis and which oligonucleotides are recognized by the RdRp.

Publications:

Guan, H. and Simon, A. E.  (2000)  Polymerization of non-template bases prior to transcription initiation at the 3' ends of templates by an RNA-dependent RNA polymerase:  An activity involved in 3'-end repair of viral RNAs.  Proc. Natl. Acad. Sci USA 97, 12451-12456.

Nagy, P. D., Carpenter, C. D., and Simon, A. E. (1997) A novel 3' end repair mechanism in an RNA virus. Proc. Natl. Acad. Sci. USA. 94, 1113-1118.

Carpenter, C. D., and Simon, A. E. (1996) In vivo repair of deletions at the 3' end of a TCV satellite RNA in vivo may involve two abortive synthesis and priming events. Virology 226, 153-160.

Carpenter, C. D. and Simon, A. E. (1996) In vivo restoration of biologically active 3' ends of virus-associated RNAs by non-homologous RNA recombination and replacement of a terminal motif. J. Virol. 70, 478-486.