Viral tRNA-like structure hijacks host ribosomes for poly(A)-independent translation

Viral tRNA-like structure hijacks host ribosomes for poly(A)-independent translation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Viral tRNA-like structure hijacks host ribosomes for poly(A)-independent translation Guoliang Lu , Liming Wan , Yuchen Chen , Ye Li , Yajie Yan , Yan Liu , Jinzhong Lin doi: https://doi.org/10.1101/2025.10.12.681957 Guoliang Lu 1 State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Zhongshan Hospital, Fudan University ; Shanghai 200438, China 2 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University ; Shanghai 200438, China 3 Center for mRNA Translational Research, Fudan University ; Shanghai 200438, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Liming Wan 1 State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Zhongshan Hospital, Fudan University ; Shanghai 200438, China 2 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University ; Shanghai 200438, China 3 Center for mRNA Translational Research, Fudan University ; Shanghai 200438, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yuchen Chen 1 State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Zhongshan Hospital, Fudan University ; Shanghai 200438, China 2 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University ; Shanghai 200438, China 3 Center for mRNA Translational Research, Fudan University ; Shanghai 200438, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ye Li 1 State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Zhongshan Hospital, Fudan University ; Shanghai 200438, China 2 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University ; Shanghai 200438, China 3 Center for mRNA Translational Research, Fudan University ; Shanghai 200438, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yajie Yan 1 State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Zhongshan Hospital, Fudan University ; Shanghai 200438, China 2 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University ; Shanghai 200438, China 3 Center for mRNA Translational Research, Fudan University ; Shanghai 200438, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yan Liu 1 State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Zhongshan Hospital, Fudan University ; Shanghai 200438, China 2 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University ; Shanghai 200438, China 3 Center for mRNA Translational Research, Fudan University ; Shanghai 200438, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jinzhong Lin 1 State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Zhongshan Hospital, Fudan University ; Shanghai 200438, China 2 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University ; Shanghai 200438, China 3 Center for mRNA Translational Research, Fudan University ; Shanghai 200438, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: linjinzhong{at}fudan.edu.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Positive-sense RNA viruses often use 3′ tRNA-like structures (TLSs) instead of poly(A) tails to capture the host translation machinery. While TLSs resemble canonical tRNAs and engage host factors, their ability to directly recruit ribosomes has remained unresolved. Here, we present cryo-electron microscopy snapshots of the histidine-accepting TLS (TLS His ) from tobacco mosaic virus bound to the 60 S subunit, the 80 S ribosome, and the 80 S -tRNA i Met initiation complex. Across these states, TLS His is consistently anchored to the L1 stalk of 60 S , even under cycloheximide treatment, yet remains dynamic on the 40 S subunit. Structural analysis shows that TLS His transitions from the E-site to an adjacent Z-site to accommodate initiator tRNA. Functional assays show that TLS His preferentially associates with ribosomal subunits over polysomes and can substitute for a poly(A) tail to promote robust cap-dependent translation. Together, these findings reveal how a viral tRNA mimic hijacks host ribosomes to promote poly(A)-independent translation. Main Text In eukaryotic cells, mRNA translation typically requires the 5′ cap and 3′ poly(A) tail to recruit translational factors, such as the eukaryotic initiation factor 4F (eIF4F) complex and the cytoplasmic poly(A)-binding protein (PABPC) 1 , 2 . The eIF4F complex recognizes the cap structure and recruits the 43 S pre-initiation complex, forming the 48 S translation initiation complex that scans the mRNA for the start codon 2 – 4 . Upon start-codon recognition, the 60 S ribosomal subunit joins to assemble the 80 S initiation complex, enabling translation to proceed 3 . The poly(A) tail, present in nearly all human mRNAs, interacts with PABPC to form a closed-loop structure with eIF4F, thereby enhancing translation initiation efficiency 5 , 6 . In contrast, many viruses have developed mechanisms to bypass cap-poly(A)-dependent translation 7 , 8 . For example, internal ribosome entry sites (IRESs) in coxsackievirus B3 (CVB3) and hepatitis C virus (HCV) bypass cap-dependent translation, while tRNA-like structures (TLSs) at the 3′ end of viral genomes replace the poly(A) tail 8 , 9 . Despite their critical roles, the exact mechanisms by which these RNA elements bypass canonical translation remain poorly understood, particularly in the case of TLSs. TLSs are remarkable RNA elements found at the 3′ ends of many positive-sense plant RNA viruses, such as tymoviruses, tobamoviruses, and bromoviruses 8 , 10 – 12 . These structures mimic tRNA functions and can hijack host translation machinery in ways that have long defied understanding since their discovery in the 1970s 13 . TLSs are classified into three major types, TLS Val , TLS His , and TLS Tyr , based on the amino acid they accept 13 – 15 . Each class adopts a distinct secondary structure that differs from tRNA, demonstrating that tRNA mimicry can occur through diverse architectural strategies 8 . Over the past decades, biochemical studies have shown their aminoacylation activity and affinity for the translation factor eEF1A 13 – 18 . Structural breakthroughs have revealed how these RNA elements fold into tRNA-like structures and interact with host translation factors. The crystal structure of the TLS Val from turnip yellow mosaic virus (TYMV) has been resolved at atomic resolution, revealing a tightly packed architecture that mimics the canonical tRNA L-shape and explaining its recognition by valyl-tRNA synthetase and eEF1A 19 . More recently, cryo-EM analyses of the TLS Tyr from brome mosaic virus (BMV) uncovered a more flexible, modular structure, likely facilitating binding by tyrosyl-tRNA synthetase and engagement with translation factors 20 . In contrast, the TLS His from tobacco mosaic virus (TMV) has long resisted structural characterization 12 . TLS His lacks the compact tertiary packing observed in TLS Val and TLS Tyr , displaying pronounced conformational heterogeneity and a tendency to aggregate 21 . Consequently, TLS His remains the last major TLS class without an atomic-level structure. Evidence suggests that TLSs exert diverse, virus-specific roles in translation. In TYMV, the TLS Val consists of an upstream pseudoknot domain (UPD) and a following compact tRNA-like domain (TLD). The UPD fine-tunes translation based on local ribosome density, while the TLD enhances translation of genomic RNA in an aminoacylation-dependent manner 22 . In TMV, the TLS His contains multiple pseudoknots (PK1-3) followed by a histidine-accepting TLD, with PK3 proposed to replace the poly(A) tail during cap-dependent translation 23 . In BMV, disruption of TLS Tyr markedly reduces translation of replication proteins, while providing TLS-containing RNA or truncated fragments in trans partially restores translation, suggesting a role for TLS in promoting translation initiation or maintaining mRNA stability 24 . The “Trojan Horse” hypothesis proposed that TLS Val could act as a pseudo-initiator tRNA, initiating translation with a valine residue in a cap-independent manner 25 , which provided an important conceptual framework for understanding TLS-mediated translation. However, direct structural evidence for ribosome recruitment by TLSs has remained elusive. In this study, we resolve this gap by capturing the TLS His from TMV in action on host ribosomes. Using a viral mini-genome mimic, we determined cryo-EM structures of the TMV TLS His bound to the 60 S subunit, the 80 S ribosome, and the 80 S -tRNAi Met initiation complex. These structures reveal that TLS His bridges the 80 S ribosome by anchoring firmly to the L1 stalk of the 60 S subunit while dynamically engaging the 40 S subunit, a binding mode distinct from both canonical tRNAs and the “Trojan Horse” model. Structural analysis shows that TLS His transits from the E-site to the Z-site, a recently defined tRNA-release intermediate, to accommodate P-site initiator tRNA. Functional assays confirm that TLS His can substitute for the poly(A) tail and support robust translation, establishing TLSs as bona fide ribosome recruitment elements. Together, these findings provide direct structural and mechanistic evidence that a viral TLS hijacks host ribosomes by exploiting the tRNA-exiting channel to stabilize initiation-competent ribosomes. Results TLS His engages host ribosomal subunits The ∼6.4 kb single-stranded TMV genome encodes a replicase and produces two subgenomic RNAs (sgRNAs) for the movement protein (MP) and the coat protein (CP) 26 . All transcripts carry a 5′ cap and a 3′ UTR containing a histidine-accepting tRNA-like structure 27 ( Figure 1A ). Whereas the 5′ cap ensures recruitment of canonical initiation factors, how the specialized 3′ architecture contributes to translation remains unresolved. Download figure Open in new tab Figure 1. TLS His -mediated translation enhancement (A) Schematic representation of the TMV genome and subgenome, each terminating in a 3′ TLS. (B) RNA pull-down with biotinylated TLS His followed by LC–MS identifies enriched host proteins, predominantly ribosomal components. Polysome profiling (C) and ribosome pelleting (D) reveal preferential TLS His association with free 40 S and 60 S subunits, with ribosomal peaks validated by mass spectrometry (Supplementary Data 1). (E–G) Translation efficiency of reporter mRNAs in WGE (E), HEK293T cells (F), and BY-2 protoplasts (G), measured by luciferase activity. (H) Covariation consensus and sequence model of TLS His variants related to the TMV TLS. We surveyed 67 viral strains (37 tobamoviruses and 30 tymoviruses) listed in the latest ICTV release and retained 28 complete 3′ UTR sequences (27 Tobamovirus , 1 Tymovirus ) that contain both the UPD and the TLD for alignment and visualization. The alignment highlights the lack of conservation in PK1 among several viruses. Abbreviations: MarMV, maracuja mosaic virus; PFMV, passion fruit mosaic virus; NeRNV, nemesia ring necrosis virus; KGMMV, Kyuri green mottle mosaic virus; CMoV, cucumber mottle virus; YTMMV, yellow tailflower mild mottle virus; ObPV, Obuda pepper virus; TMV, tobacco mosaic virus. The stop codon is shown in italic and underlined Secondary-structure consensus is annotated underlined. (I) Functional dissection of TLS His shows that both the UPD and TLD contribute to translation enhancement. To explore whether TLS His directly associates with the translation machinery, we performed a series of biochemical assays, including RNA pull-down coupled with mass spectrometry, polysome profiling, and ribosome pelleting experiments ( Figure 1B-D ). In RNA pull-downs, a biotin-labeled full-length 3′ UTR immobilized on streptavidin beads was incubated with BY-2 cell lysates, followed by LC–MS analysis. The enriched proteins were predominantly ribosomal components and translation-associated factors, indicating that TLS His engages the host translation apparatus ( Figure 1B ). Sucrose gradient polysome profiling with Cy5-labeled TMV 3′ UTR further revealed that TLS His co-sedimented mainly with free 40 S and 60 S ribosomal subunits, with little to no signal in 80 S or polysomal fractions. This suggests that TLS His primarily associates with individual subunits rather than with assembled or elongating ribosome complexes in cell extracts ( Figure 1C ). In parallel, ribosome pelleting assays using purified components demonstrated that TLS His binds directly and robustly to 40 S , 60 S , and also to 80 S ribosomes in vitro ( Figure 1D ). Together, these results provide compelling evidence that TLS His physically engages ribosomal subunits across multiple experimental systems, establishing a foundation for functional and structural analyses of how this 3′ RNA element promotes translation. TLS His promotes poly(A)-independent translation To test whether TLS His can substitute for a poly(A) tail, we generated reporter mRNAs containing the TMV 5′ and 3′ UTRs and compared translation with controls, including capped transcripts lacking the 3′ UTR, as well as capped or uncapped transcripts bearing synthetic 100-adenosine poly(A) tails. Translation assays in wheat germ extract (WGE), HEK293T cells, and tobacco BY-2 protoplasts revealed that TMV 3′ UTR-containing mRNAs supported translation as efficiently, or even more so, than polyadenylated mRNAs across all systems tested ( Figure 1E-G ). Notably, in BY-2 protoplasts, TLS His enhanced translation more than two-fold over the poly(A) tail ( Figure 1E ), highlighting its adaptation to plant-specific translational machinery. In HEK293T cells, the enhancement was more modest, consistent with previous studies in heterologous mammalian systems 23 , 28 , 29 . To examine the structural requirements for this enhancement, we created nine mutant constructs with stepwise deletions or disruptions across conserved elements of the 3′ UTR ( Figure 1H, I ). Translation assays revealed that deletions in the tRNA-like domain (TLD) had minimal impact, while removing PK3 reduced translation by ∼2-fold and removing the entire upstream pseudoknot domain (UPD) caused an ∼8-fold decrease. These results confirm that PK3 is a primary structural determinant of TLS His -mediated translation enhancement ( Figure 1I ). Notably, deletion of PK1 or PK2 led to a slight increase in translation, consistent with previous reports that PK3 in the TMV UPD is critical for replacing the poly(A) tail in cap-dependent translation 23 , 29 . Even with PK3 or UPD deletions, expression levels remained significantly higher than those of transcripts lacking the 3′ UTR, suggesting that TLS His -mediated enhancement is multifaceted. Both the upstream pseudoknots and the tRNA-like domain work together to enable the TMV 3′ UTR to replace the poly(A) tail and sustain efficient translation initiation. Cryo-EM snapshots of TLS His bound to host ribosomes To address how TLS His engages ribosomes, we attempted to resolve the structure of the ribosome-TLS His complex. Initial complexes assembled with human ribosomes yielded poor TLS His density. We therefore turned to the natural host, isolating ribosomes from Nicotiana benthamiana leaves. A synthetic mini-genome RNA containing the capped TMV 5′ UTR, ∼300 nt of coding sequence, and the full 3′ UTR efficiently assembled into ribosome complexes, aided by yeast tRNA to increase heterogeneity ( Figure 2A ). Download figure Open in new tab Figure 2. Cryo-EM structures of TLS His bound to host ribosomes (A) Experimental workflow for TLS His -ribosome complex assembly and cryo-EM grid preparation. (B) Representative 2D class averages showing RNA density within tRNA-binding clefts of 60 S and 80 S ribosomes. (C) Focused 3D classification around P- and E-sites identifies distinct classes capturing TLS His and P-/E-site tRNA occupancy. (D) Cryo-EM reconstructions of TLS His bound to 60 S (left), 80 S (middle), and 80 S with initiator tRNA (right). TLS His consistently anchors to the L1 stalk. 60 S /40 S rRNA and proteins are shown in light/dark blue and yellow/orange; TLS His and initiator tRNA in dark green and red. Inset highlights modular UPD and TLD organization, with mRNA path showing canonical P-site AUG and E-site ACA triplets matching the TMV minigenome sequence. From 17,264 micrographs, we extracted 3,455,822 particles. Two-dimensional classification revealed strong RNA densities within the tRNA-binding clefts of both 60 S and 80 S ribosomes ( Figure 2B ). Subsequent 3D classification yielded well-defined 60 S and 80 S populations. To remove free ribosome signal, particle subtraction combined with 3D classification focused on the E-site and P-site enriched three major states: TLS His bound to the 60 S subunit, the 80 S ribosome, and the 80 S with a P-site tRNA ( Figure 2C , Figure S1). Local refinements centered on TLS His and the L1 stalk yielded three distinct structures at 3.86 Å (60 S -TLS His ), 2.96 Å (80 S -TLS His ), and 4.88 Å (80 S -tRNA-TLS His ) ( Figure 2D and Figure S1). In all structures, the TLS His adopts a bipartite T-shaped architecture. The tRNA-like domain folds into a compact L-shape that is buried within the 60 S E-site, while the upstream pseudoknot domain projects laterally outward from the E-site ( Figure 2D ). In the 80 S -tRNA-TLS His complex, the P-site tRNA was unambiguously identified as initiator tRNA i Met , with clear modification densities ( Figure 2D ). Within the mRNA channel, the AUG start codon paired with the anticodon of tRNA i Met , whereas an upstream ACA trinucleotide stacks in the E-site of the 40 S neck without pairing to TLS His . Both features precisely match the TMV 5′ UTR encoded in the synthetic mini-genome ( Figure 2D ). Structural architecture of TLS His Based on the atomic model and structure-guided sequence alignment, TLS His spans 179 nucleotides (residues 26-204) within the 3′ UTR, preceded by a 25-nucleotide linker located immediately downstream of the viral stop codon (UGA). TLS His comprises two major domains, the UPD (residues 26-98) and the TLD (residues 99-204) ( Figure 3A ). The UPD contains three sequential pseudoknots, PK1 to PK3, while the TLD adopts a tRNA-like fold. Cryo-EM map enabled model building from PK2 to the TLD (residues 48-204), covering ∼87.7% of the TLS His . Notably, PK1 is poorly conserved. In several viruses, such as Maracuja mosaic virus (MarMV) and Nemesia ring necrosis virus (NeRNV), the stop codon is immediately followed by a sequence corresponding to PK2 ( Figure 1H ), suggesting PK1 is dispensable in some lineages. Download figure Open in new tab Figure 3. Structural architecture of TLS His (A) Domain organization and secondary structure of TLS His . UPD comprises PK1 (disordered, gray), PK2 (dark green), and PK3 (green). TLD contains the acceptor (purple), T (pink), D (light green), and anticodon (olive) arms, with the anticodon arm further subdivided into ASL1, ASL2, and a bulge. (B) Superposition of TLS His and canonical tRNA His , highlighting D-arm and elbow rearrangements. D/T-loop interactions mimic the canonical tRNA His fold. (C) Side view of TLS His and tRNA His with the discriminator base pair shown as sticks within the rectangle. TLS His contains an A187:C201 wobble base pair that spatially mimics the canonical G-1:C73 discriminator pair found in tRNA His . (D-E) Overview of the ASL (D) and UPD (E) domains. ASL and PKs are shown in cartoons overlaid on the surface. The TLD forms a L-shaped fold (∼70 Å in length), broadly mimicking the canonical tRNA geometry ( Figure 3B, C ). Its acceptor arm includes a 3′ pseudoknot resembling TLS Val from TYMV but one base pair shorter (Figure S2). Unlike TYMV, TLS His lacks long-range D-loop interactions, likely reducing structural stability. The discriminator base C201 forms contacts with A187, anchoring the acceptor terminus ( Figure 3C ). The T-arm closely mirrors canonical tRNA His , while the D-arm is replaced by a three-way pseudoknot ( Figure 3B ). Essential D/T-loop interactions are preserved: G108 (G18 in tRNA His ) intercalates into the T-loop, and G109 (G19) base pairs with C173 (C56), stabilizing the elbow 30 ( Figure 3B ). The anticodon stem is interrupted by a prominent bulge, forming two helices (ASL1 and ASL2) with ASL2 nearly parallel to the acceptor arm ( Figure 3C, D ). This arrangement positions the D-pseudoknot as a central hub, likely nucleating the overall fold of TLS His fold. Consistent with this role, mutations that disrupt the D/T-loop pseudoknot in related TLS systems severely impair aminoacylation activities 12 . The flexible UPD limited resolution to PK2 and PK3, which extend ∼55 Å from the D-loop pseudoknot ( Figure 3E ). These pseudoknots are compactly stacked in sequence, forming a continuous projection outward from the TLD. TLS His in action on host ribosomes TLS His engages host ribosomes by anchoring directly to the L1 stalk of the large subunit ( Figure 4A ). The ribosomal L1 stalk, a mobile element that guides tRNA movement during translocation, alternates between open and closed states 31 , 32 ( Figure 4B ). TLS His exploits this structural plasticity by coordinating L1 stalk dynamics and repositioning relative to the P-site. Download figure Open in new tab Figure 4. Structural basis of TLS His -ribosome engagement (A) Overview of TLS His complexed with 60 S , 80 S , and 80 S -tRNAi Met . TLS His anchors to the L1 stalk across all complexes; In the 60 S -TLS His and 80 S -TLS His complexes, ASL2 extends toward the P-site, while shifting outward upon initiator tRNA binding. (B) Cartoon illustration of L1 stalk in closed, half-closed, and open conformations, corresponding to TLS His -60 S , TLS His -80 S , TLS His -80 S -tRNAi Met complexes. (C) TLS His engages 60 S L1 stalk via D-arm; comparison with canonical tRNA (PDBID: 6gz3). (D) Close-up views of the terminal CCA pocket on the 60 S subunit and TLS His -uS7 contacts on the 40S subunit in the 80 S -TLS His complex. A204 is sandwiched between 25 S rRNA bases G2796 and G2797, whereas C203 stacks against the aromatic side chain of eL42-Y43. (E) Comparison of 40S binding patterns between 80 S -TLS His and 80 S -TLS His -tRNAi Met . In the absence of tRNA i Met , TLS His engages uS7 through both the PK3 and the bulge region of ASL. Upon initiator tRNA binding, TLS His shifts outward, and the interaction is re-established primarily via the backbone of the bulge and the turn between α6-7. (F) Open-book view of the TLS His -L1 stalk interface, highlighting three contact networks (dashed circle) with interacting residues shown as sticks. In the 60 S -TLS His complex, L1 stalk adopts a closed conformation, with the TLD deeply anchored in the E-site. The terminal A204 inserts into the 28 S rRNA while the A203 stacks against Tyr43 of eL42 ( Figure 4D ). The rearranged elbow engages the canonical L1 stalk platform, the G109– C173 base pair stacks against the noncanonical G2457-A2489 pair of L1 rRNA, U107 inserts into the minor groove of the L1 helix, and the sugar of G151 stacks against U2456 ( Figure 4C, F ). Together, these interactions enlarge the interface to ∼420 Å 2 compared with ∼280 Å 2 in canonical tRNAs 32 , 33 ( Figure 4C ). By contrast, TLS His exhibits conformational flexibility on the 40 S subunit. Stable binary 40 S -TLS His complexes could not be captured, but reconstruction of 80 S -TLS His complex reveals TLS His contacts with two patches on uS7. The loop between α6 and α7 on the 40 S head engages the PK3 backbone, while the α7 helix at the neck interacts with the bulge loop of the ALS, orienting ASL2 toward the P-site ( Figure 4D, E ). The TLS His elbow remains anchored to the L1 stalk, and its terminal CCA stays positioned in the E-site, as in the 60 S complex. Meanwhile, ASL2 protrudes into the P-site, sterically preventing initiator tRNA binding. In the 80 S -tRNAi Met -TLS His complex, the ribosome adopts a classical nonrotated state with the tRNA in P-site 34 . The L1 stalk shifts to an open conformation, and the bulge of TLS His slides along uS7 to replace PK3 interactions ( Figure 4D, E ). The bulge-uS7 interaction recapitulates the structural configuration observed for Z-site tRNA stabilized by anticodon-eS25 contacts, described below. TLS His rotates by ∼17° relative to the 60 S structure and ∼9° relative to the free 80 S (Figure S3), allowing ASL2 to vacate the P site while stabilizing initiator tRNA. By toggling between closed, half-closed, and open states, TLS His couples L1 stalk dynamics with P-site rearrangements, thereby facilitating the early steps of translation initiation. TLS His remains bound despite E-site occupancy TLS His can likely be aminoacylated with histidine in vivo , yet our structures show deacylated TLS His bound to the ribosomal E-site with its acceptor arm, including the CCA terminus, positioned similarly to a classical deacylated tRNA. To test if aminoacylation influences TLS His binding, we instead employed the antibiotic cycloheximide (CHX) to sterically block the E-site, taking advantage of its well-characterized ability to prevent deacylated tRNA from occupying the E-site of ribosomes 35 . Polysome profiling performed in the presence of CHX revealed that TLS His continued to co-sediment with 40 S and 60 S fractions, indicating that its association with large ribosomal subunits does not require classical insertion of the CCA end into an unoccupied E-site ( Figure 5A ). Ribosome pelleting assays further confirmed that CHX had no detectable impact on TLS His binding to the 60 S subunit, nor did treatment with puromycin (Puro) or homoharringtonine (HHT), which targe the 60 S A-site 36 ( Figure 5B ). Download figure Open in new tab Figure 5. TLS His remains bound to 60 S in the presence of CHX (A) Polysome profiling shows CHX does not disrupt TLS His association with free 60 S subunits. (B) Ribosome pelleting confirms stable TLS His -60 S binding despite E-site occupation. (C) Comparison of CHX-bound and CHX-free TLS His aligned on 25 S rRNA (L1 stalk, residues 2454-2500) reveals an outward rotation of ∼23°. (D) Cryo-ET structures capture deacylated tRNA in the intermediate Z-site of the human 80 S ribosome (PDBID: 9azs-E-tRNA; 9b0o-Z-tRNA). (E) Structural comparison of TMV TLS His with HCV and HalV IRESs highlights a shared strategy, the ribosomal tRNA exiting channel is broadly repurposed as a regulatory hub where viral RNAs stabilize initiation-competent ribosomal states (PDBID: 4ujc-HCV-IRES; 7a01HalV-IRES). Viral RNA elements or E/Z-site tRNA are shown in dark green, P-site tRNA in red, A-site tRNA in orange, and eEF1A/eIF5B in cyan. (F) Comparison of E-site and Z-site tRNAs with TLS His . The closed TLS His -60 S complex resembles an E-site tRNA, whereas the open conformation captured with 60 S --CHX and 80 S -tRNA i Met mimics a Z-site tRNA. (G) Comparison of viral IRESs with E/Z-site tRNAs. The HCV type III IRES engages the E-site via domain IIb, while the HalV IRES spans from the Z-site to the P-site. P-, E-, and Z-site tRNAs are shown in red, white, and green, and IRES domains in green cartoon overlaid on the surface. To explore this interaction structurally, we determined the cryo-EM structure of the TLS His with 60 S under CHX treatment (Figure S5). The resulting map clearly resolved CHX occupying the E-site, engaging conserved 25 S rRNA nucleotides and overlapping the position normally occupied by the A76 of a deacylated tRNA, consistent with its established mechanism of E-site blockade (Figure S4A) 35 . Remarkably, despite this blockade, TLS His remained stably anchored to the L1 stalk via its elbow region. This partial release of anchoring allowed the L1 stalk-TLS His module to swing outward by ∼23° relative to the CHX-free 60 S complex, and ∼6° relative to the 80 S -TLS His -tRNA i Met complex ( Figure 5C , S4C). This outward repositioning shifts ASL2 away from the P-site, eliminating potential steric clashes with initiator tRNA during subunit joining. Superimposition of the L1 stalk revealed that its interface with the TLS His elbow was essentially unchanged (Figure S4B). Only the terminal CA dinucleotide of TLS His became displaced and unresolved, reflecting loss of stable CCA stacking. Comparison with ribosome-bound Z-site tRNA 37 – 41 , recently described as release intermediates, reveals a striking parallel. Translation inhibition by CHX leads to robust accumulation of tRNA at the Z-site, closely resembling our observations 41 . Thus, TLS His exploits a similar site, but instead of marking release, it stabilizes a configuration that primes translation initiation ( Figure 5D, F ). This strategy also mirrors other viral RNAs that utilize the E-site to promote initiation. Type III and IV IRESs, such as those in HCV 42 , halastavi árva virus (HalV) 43 , and cricket paralysis virus (CrPV) 44 , extend structural domains into the E-site to either position the initiator tRNA or bypass it entirely via pseudoknot-mediated decoding, often in coordination with the L1 stalk ( Figure 5E, G ). These viral RNAs repurpose the E-site to facilitate initiation, leveraging structural mimicry and ribosomal anchoring to control translation. Together, these findings demonstrate that TLS His binding does not rely on canonical CCA insertion. Instead, steric blockade of the E-site, whether by aminoacylation or CHX, shifts E-site TLS His into a Z-tRNA-like configuration that favors 60 S joining and 80 S assembly. Converging strategies across TLSs and viral IRESs suggest that the ribosomal E-site, traditionally viewed as an exit pathway for deacylated tRNAs, has been broadly repurposed as a regulatory hub for viral RNAs to stabilize initiation-competent ribosomal states. A model for TLS His -driven poly(A)-independent translation Our combined biochemical and structural data support a model in which TLS His enables TMV RNAs to achieve robust translation without a poly(A) tail by directly recruiting and positioning ribosomal subunits ( Figure 6 ). Download figure Open in new tab Figure 6. Model of TLS His -mediated translation enhancement. Schematic illustrating TLS His (color-coded as in Figure 3) promoting cap-dependent translation without a poly(A) tail. TLS His can associate with both ribosomal subunits. When bound to the 40 S subunit (a), TLS His facilitates recruitment of the 60 S subunit (c), enabling assembly of an initiation-competent 80 S ribosome. Alternatively, TLS His can first engage the 60 S E-site (b), where its TLD anchors to the E-site, thereby pre-positioning the large subunit for joining with the 48 S pre-initiation complex to form the 80 S initiation complex. (2) Once the 80 S initiation complex is assembled, TLS His is displaced upon A-site tRNA accommodation and nascent peptide elongation. (3) The released TLS His is then available to capture new 40 S or 60 S subunits, supporting successive rounds of initiation. The TMV genome carries a 5′ cap and thus initiates translation via the canonical cap-dependent pathway. Functional assays demonstrate that TLS His can effectively substitute for a poly(A) tail in promoting cap-dependent translation, with the UPD, particularly PK3, playing a role analogous to the poly(A)-PABP-eIF4F system that circularizes host mRNAs and stabilizes initiation complexes. In place of this typical host strategy, TMV employs TLS His as an RNA-encoded mechanism to drive translation. Our cryo-EM structures provide a mechanistic basis for this substitution. TLS His stably anchors to the 60S subunit via its TLD, inserting into the E-site and extensively engaging the L1 stalk. This association likely enables TMV RNAs to sequester free 60 S subunits, ensuring a local pool of large subunits that are readily available for rapid initiation on viral transcripts. Concurrently, TLS His positions PK3 to contact the 40 S head, particularly via phosphate backbone interactions with uS7, effectively bridging the large and small subunits. This arrangement pre-positions a 60 S subunit adjacent to the scanning 48 S complex on the TMV 5′ UTR, lowering the energetic barrier for subunit joining and accelerating assembly of the 80 S initiation complex. This model is further supported by our polysome profiling data, which show enrichment of TLS His with free 40 S and 60 S subunits but little association with 80 S or heavy polysome fractions. Such a distribution suggests that TLS His binding to 80 S ribosomes is transient, consistent with a scenario in which TLS His promotes subunit joining but disengages once elongation commences. After each round of initiation, TLS His would then be free to bind another 60 S and/or 40 S subunit, perpetuating the cycle and driving successive rounds of translation initiation on the same viral RNA. The mobility of TLS His on the ribosome, anchored through interactions with the L1 stalk on the 60 S and phosphate backbone contacts with the 40 S , likely facilitates this process. By loosely bridging ribosomal subunits, TLS His remains dynamic enough to accommodate incoming initiator tRNAs. During elongation, displacement from the Z-site may occur as the deacylated P-site tRNA translocates into the E-site, freeing TLS His to capture additional 60 S subunits for new initiation events. Through this mechanism, TLS His not only replaces the classical poly(A)-mediated recruitment machinery but also actively organizes ribosomal subunits, endowing the TMV TLS element with the capacity to enhance translation in cis . Discussion Our study provides the first high-resolution structural characterization of a viral TLS bound to host ribosomes. This not only yields the first ribosome-engaged structure of TLS His , but also completes the structural landscape of the three major viral TLS classes: TLS Val19 , TLS Tyr20,45 , and TLS His , each now defined in molecular detail (Figure S5). Beyond its previously known interaction with aminoacyl-tRNA synthetases, TLS His is here visualized directly in complex with ribosomal subunits, establishing a new paradigm for TLS function. Together with our biochemical assays, these findings illuminate how TLS elements can act directly in translation, extending their established roles in replication and genome packaging. TLS His exhibits a divergent adaptation of the canonical tRNA fold. Although it retains the overall L-shaped geometry, it incorporates distinctive local features absent from both canonical tRNAs and other viral TLSs. The most notable is the replacement of the D-arm with a three-way pseudoknot junction 46 , in which a conserved GG dinucleotide engages the T-loop to mimic the canonical D/T-loop pairing of authentic tRNA His12 . Moreover, the anticodon arm is disrupted by a bulge that distorts the anticodon stem loop, raising questions about how these deviations affect recognition by HisRS. Unlike canonical tRNA His , which typically requires a G-1:C73 base pair and a free monophosphate at G-1 for efficient recognition 30 , TLS His exhibits an A187:C201 pairing. Earlier biochemical data showed that mutation of G-1 to A-1 does not abolish aminoacylation 30 . These findings suggest that HisRS recognition is more permissive than previously appreciated, and future structural studies of the TLS His -HisRS complex will be essential to understand how such noncanonical features are accommodated. Our data reveal that TLS His replaces the classical poly(A)-PABPC-eIF4F circuit through a modular RNA mechanism. Functional assays showed that the UPD, particularly PK3, plays a role analogous to a poly(A) tail in bridging events that promote translation. This conclusion aligns with previous deletion analyses that established PK3 as critical for poly(A)-independent enhancement 23 , 29 . Our structures now provide a mechanistic basis by showing PK3 contacts with uS7 on the 40 S head, positioning TLS His for productive engagement with the P-site. This modular architecture recalls SERBP1, recently implicated in bridging ribosomal subunits in vivo , and underscores the evolutionary flexibility of 3′ RNA modules 41 . Strikingly, related tobamoviruses such as hibiscus latent Singapore virus achieve similar outcomes using internal poly(A) tracts instead of structured UPDs 47 , illustrating parallel evolutionary solutions for maintaining translation without a canonical poly(A) tail. Across all captured complexes, TLS His -60 S , TLS His -80 S , TLS His -80 S -tRNA i Met , and 60 S -TLS His under CHX treatment, the TLS consistently anchors within the E-site while sampling closed, half-closed, and open stalk conformations. This dynamic engagement mirrors the natural motions of the L1 stalk during tRNA translocation, but here TLS His exploits them to facilitate initiation. The TLS His elbow establishes an unusually extensive interface with the L1 stalk 32 , 36 , and this interaction persists even when the CCA end is destabilized, suggesting that aminoacylation is dispensable for ribosome binding. Strikingly, this conformation parallels the recently described “Z-site” tRNAs 38 – 41 . The release intermediates positioned adjacent to the E-site and terminal A76 no longer stacks within 28S rRNA 41 . Both the displaced CCA terminus and the relative orientation resemble TLS His , suggesting that the site normally sampled by exiting tRNAs is exploited by viral TLSs to stabilize a configuration that primes initiation. Several viral RNAs similarly exploit the ribosomal E-site. The HCV IRES inserts its IIb and IIf domains into the E-site to position initiator tRNA 42 , while the HalV IRES binds the 80 S directly and uses a downstream pseudoknot to bypass canonical initiation 43 . Other examples include cap-independent translation enhancers (CITEs), such as the turnip crinkle virus 3′ CITE, which interacts with the 60S subunit to stimulate initiation 48 . TLS His thus joins a broader repertoire of structured viral RNAs that use the E-site as a regulatory hub. From an evolutionary perspective, direct ribosomal recruitment by TLS His likely provides TMV with a translational advantage. By pre-binding free 60 S subunits and bridging them to the 5′ engaged 40 S complex, TLS His may accelerate 80 S assembly and thereby favor the efficient translation of viral proteins. Moreover, association with 60 S could bias ribosome availability toward viral RNAs, particularly under cellular stress or host translation shutoff. Such strategies exemplify a general evolutionary principle: structured RNA elements evolve to co-opt host translation machinery, ensuring robust viral protein synthesis even in adverse environments. Collectively, our findings establish TLS His as a sophisticated RNA-based alternative to poly(A)-mediated translation enhancement, illustrating how viruses harness tRNA mimicry not only for replication and encapsulation but also to directly remodel host translation. Whether this strategy of direct 60 S recruitment extends to other TLS classes, such as TLS Val or TLS Tyr , remains an open question deserving further study. More broadly, these insights highlight opportunities to engineer synthetic mRNAs that incorporate TLS modules to achieve stable, high-efficiency translation, a concept with potentially transformative applications in mRNA vaccine and therapeutic design. Author contributions J.L. conceived and supervised the project. Y.C., L.W., and G.L. prepared RNA constructs. G.L., Y.C., and Y.Li. purified tobacco ribosomes. G.L. performed ribosome pelleting, polysome profiling, and RNA pull-down assays with assistance from J.L., Y.C., Y.Li., J.Y., Y.Liu., and L.W. L.W. conducted the HEK293T cell-based translation assays, while G.L. carried out the wheat germ extract and tobacco protoplast translation experiments. Cryo-EM grids were prepared by G.L. and Y.C. Cryo-EM data processing was performed by J.L. and G.L. Structural models were built and analyzed by J.L. and G.L. Data interpretation and manuscript writing were led by J.L. and G.L., with input from all authors. All authors reviewed and approved the final manuscript. Competing interests The authors declare no competing interests. Data availability The coordinates and cryo-EM density maps for TLS His -60 S , TLS His -80 S , TLS His -80 S -tRNA i Met , and CHX-TLS His -60 S have been deposited under PDB and Electron Microscopy Data Bank accession codes 9VTG and EMD-65327; 9VTI and EMD-65329; 9VTH and EMD-65328; 9VTJ and EMD-65330, respectively. Acknowledgements We thank Dr. Jinbiao Ma for valuable discussions, and Li Li for technical assistance. We are grateful to the Cryo-Electron Microscopy Core Facility of the School of Life Sciences, Fudan University, and to Dejian Zhou for support with cryo-EM data collection. We also thank Chuanwei Yang for guidance on Nicotiana benthamiana cultivation, Guohui Xie and Yijing Zhang for providing the tobacco BY-2 cell line, and Lin Huang for assistance with LC-MS analyses. This work was supported by the National Key Research and Development Program of China (Grants 2023YFC2604303 and 2023YFC2306404 to JL), the National Natural Science Foundation of China (Grants 32130063, 32471349, and 32371352 to JL), the Shanghai Municipal Science and Technology Commission (Grant 24HC2810600 to JL), and the Innovation Program of the Shanghai Municipal Education Commission (Grant 2021-01-07-00-07-E00074 to JL) and the Zhangjiang mRNA Innovation and Translation Center. We sincerely acknowledge all sources of funding. Funder Information Declared National Key Research and Development Program of China , 2023YFC2604303 , 2023YFC2306404 National Natural Science Foundation of China , 32130063 , 32471349 , 32371352 Shanghai Municipal Science and Technology Commission , 24HC2810600 Innovation Program of the Shanghai Municipal Education Commission , 2021-01-07-00- 07-E00074 References 1. ↵ Brito Querido , J. , Díaz-López , I. , and Ramakrishnan , V. ( 2024 ). 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Share Viral tRNA-like structure hijacks host ribosomes for poly(A)-independent translation Guoliang Lu , Liming Wan , Yuchen Chen , Ye Li , Yajie Yan , Yan Liu , Jinzhong Lin bioRxiv 2025.10.12.681957; doi: https://doi.org/10.1101/2025.10.12.681957 Share This Article: Copy Citation Tools Viral tRNA-like structure hijacks host ribosomes for poly(A)-independent translation Guoliang Lu , Liming Wan , Yuchen Chen , Ye Li , Yajie Yan , Yan Liu , Jinzhong Lin bioRxiv 2025.10.12.681957; doi: https://doi.org/10.1101/2025.10.12.681957 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Molecular Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13894) Bioinformatics (41951) Biophysics (21456) Cancer Biology (18594) Cell Biology (25515) Clinical Trials (138) Developmental Biology (13380) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24322) Genetics (15612) Genomics (22510) Immunology (17737) Microbiology (40400) Molecular Biology (17183) Neuroscience (88619) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)