Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

Abstract

A template-dependent RNA polymerase has been used to determine the sequence elements in the 3 untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3-terminal 4 nucleotides of domain D1 indicated the importance of the 3-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3 untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity. Tobacco mosaic virus (TMV) is the type species of theTobamovirus genus of plant viruses. Its positive-sense, singlestranded RNA genome encodes four proteins (reviewed in references 3, 4, and 8). The 126-kDa protein, encoded by a 5Ј-proximal open reading frame (ORF), is translated from the genomic RNA. It has a domain in its N-terminal region with amino acid sequence motifs typical of methyltransferases, which is believed to be involved in the synthesis of the 5Ј cap structure, and a domain in its C-terminal region with sequences typical of RNA helicases. The 183-kDa protein is translated by readthrough of a stop codon at the end of the ORF for the 126-kDa protein. In addition to the two domains which it shares with the 126-kDa protein, the 183-kDa protein has a domain in its C-terminal region with amino acid sequence motifs typical of RNA-dependent RNA polymerases (RdRp). Both the 126-and 183-kDa proteins, which have been detected in isolated TMV replication complexes, are needed for efficient replication of TMV RNA. Recently a purified TMV RdRp was shown to contain a 126-kDa/183-kDa protein heterodimer (48). The role in TMV RNA replication of the excess 126-kDa protein which is produced in vivo is not known. TMV RNA also encodes a 30-kDa cell-to-cell movement protein and a 17.5-kDa capsid protein, which are translated from different 3Ј-coterminal subgenomic mRNAs.Replication of TMV RNA takes place in two stages: synthesis of a minus strand using the virus plus-strand RNA as a template is followed by synthesis of progeny ...

Replication of tobamovirus RNA

“…37) Osman et al have demonstrated that the tRNA-like structure is important for in vitro minus-strand RNA synthesis by the template-dependent ToMV RdRp. 38) Furthermore, deletions of the 3'-terminal sequences or the addition of extra non-viral sequences results in the loss or reduction of in vivo infectivity of tobamovirus RNA, 4),5) which indicates the importance of intact 3'-terminal structures. However, a recent study with Turnip yellow mosaic virus, which belongs to the Tymovirus genus in the alpha-like virus superfamily, suggests that aminoacylation of the genomic RNA itself is not an absolute requirement for RNA replication.…”

Comparison and functional implications of the 3D architectures of viral tRNA-like structures

“…The roles each play during infection vary, but in general, these include regulation/enhancement of translation, replication of the genome, fidelity of the 39 end of the viral RNA, encapsidation, viral RNA genome stability, and thus overall virus amplification. Because they lie at the 39 end of the viral genome, TLSs are among the first sequences to be replicated by the RNAdependent RNA polymerases (RDRPs) and can contain promoter sequences (Dreher et al 1984;Singh and Dreher 1997;Osman et al 2000); thus they could regulate the switch between negative strand synthesis and translation. In addition, one study proposes that the valylated TYMV TLS delivers its amino acid to the ribosome to initiate translation of a viral open reading frame in cis; this model remains controversial (Barends et al 2003;Matsuda and Dreher 2007).…”

Identification of a Region of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replication Proteins Which Binds Specifically to the Viral 3′-Terminal tRNA-Like Structure

“…Regions which were identified as being important for negative-strand synthesis in vitro included the 3Ј-terminal CA sequence, domains D1 (equivalent to a tRNA acceptor arm), D2 (similar to a tRNA anticodon arm), and D3 (an upstream pseudoknotted region), and a central core region C which connects domains D1, D2, and D3. Domain D2 and the central core region C were shown to be the most important elements for binding of the TMV RdRp complex to the viral 3Ј-TR (22).…”

“…The 183-kDa protein, which is translated as a result of ribosomal readthrough of a termination codon at the end of the open reading frame for the 126-kDa protein, additionally has a domain typical of RNA-dependent RNA polymerases (RdRps) (14). The 35-kDa cell-to-cell movement protein and the 17.5-kDa coat protein are translated from 3Ј-coterminal subgenomic RNAs.The 3Ј-terminal region (3Ј-TR) of TMV RNA can be folded into a tRNA-like structure and adjacent upstream pseudoknots (7, 31) which have been shown to be important for TMV RNA replication in vivo (3, 28) and negative-strand RNA synthesis in vitro (22,33). Regions which were identified as being important for negative-strand synthesis in vitro included the 3Ј-terminal CA sequence, domains D1 (equivalent to a tRNA acceptor arm), D2 (similar to a tRNA anticodon arm), and D3 (an upstream pseudoknotted region), and a central core region C which connects domains D1, D2, and D3.…”

“…The 3Ј-terminal region (3Ј-TR) of TMV RNA can be folded into a tRNA-like structure and adjacent upstream pseudoknots (7, 31) which have been shown to be important for TMV RNA replication in vivo (3, 28) and negative-strand RNA synthesis in vitro (22,33). Regions which were identified as being important for negative-strand synthesis in vitro included the 3Ј-terminal CA sequence, domains D1 (equivalent to a tRNA acceptor arm), D2 (similar to a tRNA anticodon arm), and D3 (an upstream pseudoknotted region), and a central core region C which connects domains D1, D2, and D3.…”