8-Hydroxyguanine in a mutational hotspot of the c-Ha-ras gene causes misreplication, 'action-at-a-distance' mutagenesis and inhibition of replication

Mutations in particular codons of c-Ha-ras have a strong activating potential, and an activated ras oncogene has been found in a number of human cancers. Using fragments of the human c-Ha-ras gene containing 8-hydroxyguanine (8-OH-G) in codon 12, we provide evidence for highly complex biochemical events leading to activation of the oncogene. Replication with DNA polymerases α (Polα) and β (Polβ) led to misincorporation of dAMP, while DNA polymerase η (Polη) caused additional insertion of dGMP. For the first time we report an ‘action-at-a-distance’ mutagenic effect for Polη. Replication catalyzed by this enzyme resulted in misincorporating dAMP, dTMP and dGMP opposite non-oxidized guanine 3′-flanked by 8-OH-G. Interestingly, two adjacent 8-OH-G residues greatly relaxed the specificity of Polη, which in this system was able to incorporate all four nucleotides. Moreover, two adjacent 8-OH-G residues completely blocked Polα and strongly inhibited Polβ, whereas Polη was entirely resistant to this inhibition. These results suggest an important role for Polη in inducing hypermutability in codon 12. Our observations are important for understanding the consequences of 8-OH-G being positioned within the mutational hot spots of oncogenes, the outcome of which appears to be relatively complex even in minimal in vitro systems.     

Influence of local sequence context on damaged base conformation in human DNA polymerase ι: molecular dynamics studies of nucleotide incorporation opposite a benzo[a]pyrene-derived adenine lesion

Human DNA polymerase ι is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions in both near error-free and error-prone bypass. With undamaged templating purines polymerase ι normally favors Hoogsteen base pairing. Polymerase ι can incorporate nucleotides opposite a benzo[a]pyrene-derived adenine lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. We performed molecular modeling and molecular dynamics simulations to elucidate the structural basis for lesion bypass. Our results suggest that hydrogen bonds between the benzo[a]pyrenyl moiety and nearby bases limit the movement of the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5′-CAGA*TT-3′ sequence. This facilitates correct incorporation of dT via a Watson−Crick pair. In a 5′-TTTA*GA-3′ sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA*, there is also an opportunity for increased misincorporation of dGTP. These results expand our understanding of the versatility and flexibility of polymerase ι and its lesion bypass functions in humans.     

Thermodynamic stability of base pairs between 2-hydroxyadenine and incoming nucleotides as a determinant of nucleotide incorporation specificity during replication

We investigated the thermodynamic stability of double-stranded DNAs with an oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two different sequence contexts (5′-GA*C-3′ and 5′-TA*A-3′, A* represents 2-OH-Ade). When an A*–N pair (N, any nucleotide base) was located in the center of a duplex, the thermodynamic stabilities of the duplexes were similar for all the natural bases except A (N = T, C and G). On the other hand, for the duplexes with the A*–N pair at the end, which mimic the nucleotide incorporation step, the stabilities of the duplexes were dependent on their sequence. The order of stability is T > G > C >> A in the 5′-GA*C-3′ sequences and T > A > C > G in the 5′-TA*A-3′ sequences. Because T/G/C and T/A are nucleotides incorporated opposite to 2-OH-Ade in the 5′-GA*C-3′ and 5′-TA*A-3′ sequences, respectively, these results agree with the tendency of mutagenic misincorporation of the nucleotides opposite to 2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*–N base pair may be an important factor for the mutation spectra of 2-OH-Ade.     

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

N²,3-Ethenoguanine (N²,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N²,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N²,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N²,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N²,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N²,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N²,3-ϵG.     

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo⁻ fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo⁻.     

Doublet preference and gene evolution

Summary Doublet preference analysis was carried out on coding and noncoding regions ofEscherichia coli, Saccharomyces cerevisiae, and human mitochondrial and nuclear DNA. The preference pattern in 1–2 and 2–3 doublets inE. coli andS. cerevisiae correlated with that in noncoding regions. The 3-1 doublet preference inE. coli genes with low optimal codon frequency and inS. cerevisiae genes also showed a correlation with each of their noncoding doublet preference. A mechanism to explain these double preference correlations in doublet preference is presented: mutational biases, the origin of the noncoding region doublet preference, evolved so as to maintain the 1–2 and 2–3 doublet preference, which is determined by codon usage. These biases then acted on the 3-1 doublet, which was almost free of coding constraints, resulting in a similar preference in this doublet.     

Translesion synthesis by yeast DNA polymerase ζ from templates containing lesions of ultraviolet radiation and acetylaminofluorene

In the yeast Saccharomyces cerevisiae, DNA polymerase ζ (Polζ) is required in a major lesion bypass pathway. To help understand the role of Polζ in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polζ performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3′ T, and predominantly A opposite the 5′ T. Purified yeast Polζ predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. Furthermore, yeast Polζ catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3′ T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3′ T of the TT (6-4) photoproduct. In contrast, the 3′ T of a cis–syn TT dimer completely blocked purified yeast Polζ, whereas the 5′ T was readily bypassed. These results support the following dual-function model of Polζ. First, Polζ catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polζ.     

Synthesis and properties of oligonucleotides containing 5-formyl-2′-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2′-deoxycytidine

Oligodeoxynucleotides (ODNs) containing 5-formyl-2′-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2′-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C·G→T·A and the transversion mutation C·G→A·T during DNA synthesis.     

Translation Initiation at the CUU Codon Is Mediated by the Internal Ribosome Entry Site of an Insect Picorna-Like Virus In Vitro

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3′-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3′ boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5′ terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5′ part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.     

Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae

In-frame stop codons normally signal termination during mRNA translation, but they can be read as ‘sense’ (readthrough) depending on their context, comprising the 6 nt preceding and following the stop codon. To identify novel contexts directing readthrough, under-represented 5′ and 3′ stop codon contexts from Saccharomyces cerevisiae were identified by genome-wide survey in silico. In contrast with the nucleotide bias 3′ of the stop codon, codon bias in the two codon positions 5′ of the termination codon showed no correlation with known effects on stop codon readthrough. However, individually, poor 5′ and 3′ context elements were equally as effective in promoting stop codon readthrough in vivo, readthrough which in both cases responded identically to changes in release factor concentration. A novel method analysing specific nucleotide combinations in the 3′ context region revealed positions +1,2,3,5 and +1,2,3,6 after the stop codon were most predictive of termination efficiency. Downstream of yeast open reading frames (ORFs), further in-frame stop codons were significantly over-represented at the +1, +2 and +3 codon positions after the ORF, acting to limit readthrough. Thus selection against stop codon readthrough is a dominant force acting on 3′, but not on 5′, nucleotides, with detectable selection on nucleotides as far downstream as +6 nucleotides. The approaches described can be employed to define potential readthrough contexts for any genome.     

Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone

2-Methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ–DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences with an internal MQ, the formation of cross-links with A and C opposite the MQ moiety was 2- to 3-fold greater than that with G and T. The yield of cross-links was more than 10-fold greater than that of breaks opposite MQ, which in turn was more than 2-fold greater than breaks at GG doublets. The yield of damage at GG doublets greatly increased for a sequence with a terminal MQ. The distribution of base damage was measured by enzymatic digestion and HPLC analysis (dAdo > dThd > dGuo > dCyd). The formation of novel products in MQ–DNA duplexes was attributed to the ability of excited MQ to generate the radical cations of all four DNA bases; thus, this photochemical reaction provides an ideal model system to study the effects of ionizing radiation and one-electron oxidants.     

Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E.coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3′- or 5′-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5′-terminus of the 3′ cleaved fragment, but is unable to remove DHU remaining on the 3′-terminus of the cleaved 5′ fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3′-terminus of a cleaved 5′ fragment, but are unable to remove DHU remaining on the 5′-terminus of a cleaved 3′ fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.     

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.     

The role of sequence context,nucleotide pool balance and stress in 2′-deoxynucleotide misincorporation in viral,bacterial and mammalian RNA

The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 10³ to 10⁵ ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections.     

Optimizing heterologous expression in dictyostelium: importance of 5' codon adaptation

Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-β gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5–17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5′-sequence of the hCG gene to the Dictyostelium ‘Kozak’-like sequence increased expression levels ~1.5-fold. Thus, using both codon optimization and ‘Kozak’ adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG.     

Twin gradients in APOBEC3 edited HIV-1 DNA reflect the dynamics of lentiviral replication

The human immunodeficiency virus (HIV) Vif protein blocks incorporation of two host cell cytidine deaminases, APOBEC3F and 3G, into the budding virion. Not surprisingly, on a vif background nascent minus strand DNA can be extensively edited leaving multiple uracil residues. Editing occurs preferentially in the context of TC (GA on the plus strand) and CC (GG) depending on the enzyme. To explore the distribution of APOBEC3F and –3G editing across the genome, a product/substrate ratio (AA + AG)/(GA + GG) was computed for a series of 30 edited genomes present in the data bases. Two highly polarized gradients were noted each with maxima just 5′ to the central polypurine tract (cPPT) and LTR proximal polypurine tract (3′PPT). The gradients are in remarkable agreement with the time the minus strand DNA remains single stranded. In vitro analyses of APOBEC3G deamination of nascent cDNA spanning the two PPTs showed no pronounced dependence on the PPT RNA:DNA heteroduplex ruling out the competing hypothesis of a PPT orientation effect. The degree of hypermutation varied smoothly among genomes indicating that the number of APOBEC3 molecules packaged varied considerably.     

Epidemiology of Doublet/Multiplet Mutations in Lung Cancers: Evidence that a Subset Arises by Chronocoordinate Events

Background

Evidence strongly suggests that spontaneous doublet mutations in normal mouse tissues generally arise from chronocoordinate events. These chronocoordinate mutations sometimes reflect “mutation showers”, which are multiple chronocoordinate mutations spanning many kilobases. However, little is known about mutagenesis of doublet and multiplet mutations (domuplets) in human cancer. Lung cancer accounts for about 25% of all cancer deaths. Herein, we analyze the epidemiology of domuplets in the EGFR and TP53 genes in lung cancer. The EGFR gene is an oncogene in which doublets are generally driver plus driver mutations, while the TP53 gene is a tumor suppressor gene with a more typical situation in which doublets derive from a driver and passenger mutation.

Methodology/Principal Findings

EGFR mutations identified by sequencing were collected from 66 published papers and our updated EGFR mutation database (www.egfr.org). TP53 mutations were collected from IARC version 12 (www-p53.iarc.fr). For EGFR and TP53 doublets, no clearly significant differences in race, ethnicity, gender and smoking status were observed. Doublets in the EGFR and TP53 genes in human lung cancer are elevated about eight- and three-fold, respectively, relative to spontaneous doublets in mouse (6% and 2.3% versus 0.7%).

Conclusions/Significance

Although no one characteristic is definitive, the aggregate properties of doublet and multiplet mutations in lung cancer are consistent with a subset derived from chronocoordinate events in the EGFR gene: i) the eight frameshift doublets (present in 0.5% of all patients with EGFR mutations) are clustered and produce a net in-frame change; ii) about 32% of doublets are very closely spaced (≤30 nt); and iii) multiplets contain two or more closely spaced mutations. TP53 mutations in lung cancer are very closely spaced (≤30 nt) in 33% of doublets, and multiplets generally contain two or more very closely spaced mutations. Work in model systems is necessary to confirm the significance of chronocoordinate events in lung and other cancers.     

Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion

REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N ²-dG, (–)-trans-anti-benzo[a]pyrene-N ²-dG and 1,N ⁶-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N ² -dG adducts and the 1,N ⁶-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass.     

Identification of cis-Acting Nucleotides and a Structural Feature in West Nile Virus 3′-Terminus RNA That Facilitate Viral Minus Strand RNA Synthesis

The 3′-terminal nucleotides (nt) of West Nile virus (WNV) genomic RNA form a penultimate 16-nt small stem-loop (SSL) and an 80-nt terminal stem-loop (SL). These RNA structures are conserved in divergent flavivirus genomes. A previous in vitro study using truncated WNV 3′ RNA structures predicted a putative tertiary interaction between the 5′ side of the 3′-terminal SL and the loop of the SSL. Although substitution or deletion of the 3′ G (nt 87) within the SSL loop, which forms the only G-C pair in the predicted tertiary interaction, in a WNV infectious clone was lethal, a finding consistent with the involvement in a functionally relevant pseudoknot interaction, extensive mutagenesis of nucleotides in the terminal SL did not identify a cis-acting pairing partner for this SSL 3′ G. However, both the sequence and the structural context of two adjacent base pairs flanked by symmetrical internal loops in the 3′-terminal SL were shown to be required for efficient viral RNA replication. Nuclear magnetic resonance analysis confirmed the predicted SSL and SL structures but not the tertiary interaction. The SSL was previously reported to contain one of three eEF1A binding sites, and G87 in the SSL loop was shown to be involved in eEF1A binding. The nucleotides at the bottom part of the 3′-terminal SL switch between 3′ RNA-RNA and 3′-5′ RNA-RNA interactions. The data suggest that interaction of the 3′ SL RNA with eEF1A at three sites and a unique metastable structural feature may participate in regulating structural changes in the 3′-terminal SL.