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

Abstract

Tobacco mosaic virus (TMV) has a 6.3-kb positive-stranded RNA genome which encodes four proteins (reviewed in references 1, 2, and 6). The 126-and 183-kDa proteins have essential replication functions and are translated from the virus genomic RNA. The 126-kDa protein has an N-terminal methyltransferase and guanylyltransferase or capping domain (14, 16) and a C-terminal helicase domain (10, 14). 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. 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 TMV RdRp complex, isolated from infected plants, consists of the 126-kDa protein, the 183-kDa protein, and a number of plant proteins (21,29,33). Until now, the protein component of the RdRp complex responsible for the specific binding to the TMV RNA 3Ј-TR was unknown. Here we have used UV cross-linking to show that a region of the 126-and 183-kDa proteins downstream of the core methyltransferase domain is responsible for binding of the RdRp to the RNA 3Ј-TR in vitro. We have also identified two aromatic amino acids in this region of the 126-and 183-kDa proteins that are essential for cross-linking to the RNA 3Ј-TR in vitro and for replication of TMV RNA in tomato protoplasts. MATERIALS AND METHODSPreparation of TMV-L RdRp. Membrane-bound viral RdRp was isolated from tomato plants infected with TMV-L by differential and density gradient centrifugation and then solubilized and purified as described by Osman and Buck (20,21).Preparation of cDNA templates and in vitro transcription. RNA t2 and the mutants described in Table 1 were synthesized by in vitro transcription using T7 RNA polymerase and a DNA template generated by PCR from cDNA clones of TMV-L RNA as described previously (22). RNAs corresponding to the 3Ј-terminal 273 nucleotides (nt) of red clover necrotic mosaic virus RNA2 and nt 2466 to 2738 of the vector LITMUS28 were synthesized by in vitro transcription using T7 RNA polymerase and templates described previously (22). RNA transcripts were purified as described in the Ambion manual and assayed spectrophotometrically.Expression of proteins in ...

Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

“…The eEF1A protein is reported to directly bind to tobamovirus replication proteins (31) as well as to pseudoknot structures in the 3Ј untranslated region of tobamovirus RNA (34). Since the tobamovirus replication proteins also bind to tobamovirus RNA (22), the observed association between eEF1A and the 180K protein may occur both through direct physical interaction and through indirect interactions mediated by the ToMV RNA. It is also possible that a fraction of eEF1A is involved in translation.…”

Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein

“…Osman and Buck (35) mapped an RNA-binding domain to amino acids 314 to 423 of the replicase protein of ToMV, and the 126k protein of TMV was shown to have methyltransferase activity, albeit with the cap as a target and S-adenosylmethionine as the methyl donor (30). It is possible that this activity could also use methylated sRNAs as methyl donors, thereby leading to their demethylation.…”

Replication of tobamovirus RNA

“…Therefore, it seems likely that this binding is essential for ToMV RNA replication. 25) Some of the replication-defective 130 K-and 180 K-defective mutants can be trans-complemented by self-replicatable helper tobamoviruses.…”