Identity of the 5′-Terminal RNA Nucleotide Sequence of the Satellite Tobacco Necrosis Virus and Its Helper Virus: Possible Role of the 5′-Terminus in the Recognition by Virus-Specific RNA Replicase

Abstract

Abstract. A pancreatic ribonuclease digest of 14C-labeled tobacco necrosis virus RNA was fractionated according to charge by column chromatography. Individual fractions were dephosphorylated with alkaline phosphatase and rechromatographed. The fraction, originally containing oligonucleotides with seven negative charges, separated into two components corresponding to five (-5) and two negative charges (-2). The -5 fraction was derived from the internal oligonucleotides while the -2 fraction must have originated from a 5'-pyrophosphorylated terminal trinucleotide. The sequence of this terminal trinucleotide was determined by column chromatography on DEAE-cellulose in a triethyl ammonium carbonate gradient, using the appropriate markers. The radioactivity chromatographed with a (ApGp)U marker. The order of the Ap and Gp was determined after ribonuclease T1 and alkaline phosphatase digestion. The radioactivity in the product chromatographed with an ApG marker. The 5'-terminus of tobacco necrosis virus RNA was therefore determined as ppApGpUp . . ., which is identical to the terminus of the RNA of its satellite virus as previously determined (J. llMol. Biol., 38, 59 (1968); Science, 160, 1452 (1968)).The 5' pyrophosphate in both viruses was probably formed by an in vivo enzymatic removal of a y-phosphate from a triphosphate, and its presence in both viruses suggested a common site of synthesis. The identity of the 5'-terminal sequences is considered not to be fortuitous and is discussed from the standpoint of their role as a recognition site for the virus-specific RNA replicase.Sequence studies of the mRNA's of viral origin'-' surprisingly failed to reveal the presence of a protein-chain initiating codon near the 5'-terminus. It must therefore be concluded that these nucleotide sequences are not part of a cistron and their biological role remains obscure. De Wachter and Fiers'showed that the 5'-terminal sequence of Q#-RNA was identical to the sequence of the "little variant"4 reported by Bishop.5 In view of the template activity of the little variant, the conservation of the 5'-terminus would suggest its possible role as a recognition site-for Q,-RNA replicase.Experiments with partly purified extracts of plants infected with tobacco necrosis virus (TNV) in our laboratory, showed that labeled nucleotides were incorporated into high molecular weight RNA under the direction of both TNV-140

Structure and function of a cap-independent translation element that functions in either the 3′ or the 5′ untranslated region

“…To determine the roles of the major internal structures in the TE, sequences encompassing each of the three stem-loops in the TE were deleted individually+ Every stem-loop deletion destroyed the function of the TE at either the 39 UTR or the 59 UTR (Fig+ 3C, mutations ⌬SL-I, ⌬SL-II, and ⌬SL-III), indicating that either the sequence or the secondary structure of these regions was critical for translation initiation+ To determine whether the sequences or the secondary structural domains contribute to TE function, the stem-loops were mutated at higher resolution+ SL-I is part of a 17-nt tract (bases 4837-4853) that is completely conserved (Wang et al+, 1997;Fig+ 2E) in all members of genus Luteovirus (with the exception of BYDV-PAV129, which has an A at position 4838), soybean dwarf virus (SDV, an unclassified member of Luteoviridae that is the closest sequence relative to BYDV; Rathjen et al+, 1994), as well as more distantly related Tobacco necrosis virus (TNV) RNA (genus Necrovirus), which also has uncapped, nonpolyadenylated RNAs (Lesnaw & Reichmann, 1970)+ All mutations that we introduced into the 17-nt tract were defective both in vitro and in vivo (Fig+ 3A,C)+ Changing GGAAA [4845][4846][4847][4848][4849] to UUUCC or to GGAA (a GNRA tetraloop) eliminated cap-independent translation (Fig+ 3A,C, mutations LI-m1 and LI-m2), indicating that the loop sequence of SL-I is important for TE function+ An interesting feature of the 17-nt conserved region is that sequence GAUCCU 4838-4843 can potentially base pair to the AGGAUC sequence located five bases from the 39 end of 18S rRNA (Fig+ 3A;Wang et al+, 1997)+ This is the location of the anti-Shine-Dalgarno sequence (mRNA-binding site) in prokaryotic 16S rRNA+ Although three of the GAUCCU 4838-4843 bases are base paired in stem I (Fig+ 3A), we can consider the possibility of RNA "breathing" or helicase-melting of SL-I+ Moreover, the arrangement of these bases resembles that of the ribosome binding site of the extremely efficiently translated coat protein gene of bacteriophage Qb, in which the three 39 bases of the sequence are in a small stem structure (Priano et al+, 1997)+ A set of mutations that disrupted potential base pairing to 18S rRNA or within Stem-I or both was constructed+ All mutations in this set abolished cap-independent translation with TE105 in the 59 UTR (Fig+ 3C, SI-m1, SI-m2, and SI-r), including one that may enhance 18S rRNA binding by disrupting SL-1 (Sl-m2)+ However, a natural variant (PAV129; Fig+ 2E), contains a G-to-A transition in the second position of the conserved 17-nt tract that allows only five bases to pair to 18S rRNA+ The 39 TE of PAV129 confers cap-independent translation on reporter genes (L+ Guo, unpubl+ observation), and virus containing the PAV129 TE is infectious (S+ Liu, pers+ comm+)+ This suggests that a Shine-Dalgarno-like interaction may not be necessary, but more definitive evidence is required to rule out such a mechanism+ Because all of the mutations in SL-1 destroyed TE activity, we conclude that its sequence is essential, but we can draw no conclusion about the role of secondary structure of SL-I+…”

“…In contrast to the above RNAs, Tobacco mosaic virus (TMV) RNA has a 59 cap but no poly(A) tail+ A pseudoknot-rich domain in the TMV 39 UTR substitutes for a poly(A) tail (Gallie & Walbot, 1990), perhaps by binding a common factor that also binds to the TMV 59 UTR in a cap-dependent manner (Tanguay & Gallie, 1996)+ In the case of Rotavirus, which also has capped, nonpolyadenylated mRNAs, viral protein NSP3A interacts with the viral 39 UTR as well as the human eIF4GI in a complex with eIF4A and eIF4E (Vende et al+, 2000)+ Thus, NSP3A substitutes for PABP in the interaction between two ends of the mRNA+ RNAs of viruses and satellite viruses in the Luteovirus (Allen et al+, 1999) and Necrovirus (Lesnaw & Reichmann, 1970;Danthinne et al+, 1993;Timmer et al+, 1993) genera, and the large Tombusviridae family (Qu & Morris, 2000) lack both a 59 cap and a 39 poly(A) tail+ However, they can be translated efficiently, owing to different translation enhancement sequences residing in their 39 UTRs (Danthinne et al+, 1993;Timmer et al+, 1993;Wang & Miller, 1995;Wang et al+, 1999;Wu & White, 1999;Qu & Morris, 2000)+ These differ from IRES in two fundamental ways: they do not confer internal ribosome entry, and they are located in the 39 UTR+ The structures and mechanism of action of these sequences are unknown+…”

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans

“…The 39 end of mRNA also participates in translation initiation (Gallie, 1991;Tarun & Sachs, 1995;Jacobson, 1996;Sachs et al+, 1997)+ The poly(A) tail interacts synergistically with the 59 cap in stimulating translation in vivo (Gallie, 1991; Tarun et al+, 1997; Preiss & Hentze, 1998)+ In viral RNAs that lack a 39 poly(A) tail, other sequences in the 39 UTR may stimulate translation (Leathers et al+, 1993)+ The RNAs of barley yellow dwarf virus (BYDV; Allen et al+, 1999) and satellite tobacco necrosis virus (STNV; Lesnaw & Reichmann, 1970) lack both a 59 cap and a poly(A) tail+ The RNAs of these viruses each contain a different sequence in the 39 UTR that confers efficient cap-independent translation on uncapped mRNA (Danthinne et al+, 1993;Timmer et al+, 1993;Wang & Miller, 1995;Wang et al+, 1997;Meulewaeter et al+, 1998)+ BYDV is in the genus Luteovirus of the family Luteoviridae+ Members of the family Luteoviridae have a single stranded, positive-sense RNA genome of 5+6 to 5+7 kb encoding about six open reading frames (ORFs) (Mayo & Ziegler-Graff, 1996;Miller, 1999)+ Viruses in the genus Polerovirus of the family Luteoviridae have a VPg linked to the 59 terminus of the genome (Mayo et al+, 1982;Murphy et al+, 1989), whereas BYDV RNA has neither a VPg (Shams-bakhsh & Symons, 1997) nor a 59 cap (Allen et al+, 1999)+ During its life cycle, BYDV produces three subgenomic RNAs (sgRNAs) that are 39 coterminal with genomic RNA (gRNA) (Fig+ 1) (Kelly et al+, 1994;Mohan et al+, 1995;Miller et al+, 1997)+ The ORFs (1 and 2) in the 59 half of genome are translated from gRNA (Wang & Miller, 1995)+ ORF 2, which encodes the RNA-dependent RNA polymerase, is translated by ribosomal frameshifting from ORF 1 to generate a 99-kDa fusion product (Di et al+, 1993)+ ORFs 3, 4, and 5 code for the coat protein, movement protein, and an aphid transmission function, respectively (reviewed by Miller, 1999)+ All three ORFs are translated only from sgRNA1 (Fig+ 1) (Brown et al+, 1996)+ ORF 4 is translated by leaky scanning (Dinesh-Kumar & Miller, 1993) and ORF 5 by in-frame readthrough of the ORF 3 stop codon (Brown et al+, 1996)+ Subgenomic RNA2 (sgRNA2) may serve as a message for ORF 6 (K...…”

The 3′ Untranslated Region of Tobacco Necrosis Virus RNA Contains a Barley Yellow Dwarf Virus-Like Cap-Independent Translation Element

“…TNV RNA has no 5Ј cap (23) and no 3Ј poly(A) tail or tRNA-like structure (24), yet it translates efficiently. Here we report that there is a BYDV-like TE in the 3Ј UTR of TNV-D RNA that confers efficient cap-independent translation.…”