Natural evidence of coronaviral 2'-O-methyltransferase activity affecting viral pathogenesis via improved substrate RNA binding.

Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune evasion. However, the K-D-K-E catalytic motif of 2'-O-MTase is highly conserved across numerous viruses, including flaviviruses, vaccinia viruses, coronaviruses, and extends even to mammals. Here, we observed a stronger 2'-O-MTase activity in SARS-CoV-2 compared to SARS-CoV, despite the presence of a consistently active catalytic center. We further identified critical residues (Leu-36, Asn-138 and Ile-153) which served as determinants of discrepancy in 2'-O-MTase activity between SARS-CoV-2 and SARS-CoV. These residues significantly enhanced the RNA binding affinity of 2'-O-MTase and boosted its versatility toward RNA substrates. Of interest, a triple substitution (Leu36 → Ile36, Asn138 → His138, Ile153 → Leu153, from SARS-CoV-2 to SARS-CoV) within nsp16 resulted in a proportional reduction in viral 2'-O-methylation and impaired viral replication. Furthermore, it led to a significant upregulation of type I interferon (IFN-I) and proinflammatory cytokines both in vitro and vivo, relying on the cooperative sensing of melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). In conclusion, our findings demonstrated that alterations in residues other than K-D-K-E of 2'-O-MTase may affect viral replication and subsequently influence pathogenesis. Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.

The impact of single-stranded RNAs on the dimerization of double-stranded RNA-dependent protein kinase PKR.

The RNA-binding protein PKR serves as a crucial antiviral innate immune factor that globally suppresses translation by sensing viral double-stranded RNA (dsRNA) and by phosphorylating the translation initiation factor eIF2α. Recent findings have unveiled that single-stranded RNAs (ssRNAs), including in vitro transcribed (IVT) mRNA, can also bind to and activate PKR. However, the precise mechanism underlying PKR activation by ssRNAs, remains incompletely understood. Here, we developed a NanoLuc Binary Technology (NanoBiT)-based in vitro PKR dimerization assay to assess the impact of ssRNAs on PKR dimerization. Our findings demonstrate that, akin to double-stranded polyinosinic:polycytidylic acid (polyIC), an encephalomyocarditis virus (EMCV) RNA, as well as NanoLuc luciferase (Nluc) mRNA, can induce PKR dimerization. Conversely, homopolymeric RNA lacking secondary structure fails to promote PKR dimerization, underscoring the significance of secondary structure in this process. Furthermore, adenovirus VA RNA 1, another ssRNA, impedes PKR dimerization by competing with Nluc mRNA. Additionally, we observed structured ssRNAs capable of forming G-quadruplexes induce PKR dimerization. Collectively, our results indicate that ssRNAs have the ability to either induce or inhibit PKR dimerization, thus representing potential targets for the development of antiviral and anti-inflammatory agents.

Structure of the SARS-CoV-2 Frameshift Stimulatory Element with an Upstream Multibranch Loop.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed -1 ribosomal frameshifting (-1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to -1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site-a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.

Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site.

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.

Tying the knot: Unraveling the intricacies of the coronavirus frameshift pseudoknot.

Understanding and targeting functional RNA structures towards treatment of coronavirus infection can help us to prepare for novel variants of SARS-CoV-2 (the virus causing COVID-19), and any other coronaviruses that could emerge via human-to-human transmission or potential zoonotic (inter-species) events. Leveraging the fact that all coronaviruses use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to replicate, we apply algorithms to predict the most energetically favourable secondary structures (each nucleotide involved in at most one pairing) that may be involved in regulating the -1 PRF event in coronaviruses, especially SARS-CoV-2. We compute previously unknown most stable structure predictions for the frameshift site of coronaviruses via hierarchical folding, a biologically motivated framework where initial non-crossing structure folds first, followed by subsequent, possibly crossing (pseudoknotted), structures. Using mutual information from 181 coronavirus sequences, in conjunction with the algorithm KnotAli, we compute secondary structure predictions for the frameshift site of different coronaviruses. We then utilize the Shapify algorithm to obtain most stable SARS-CoV-2 secondary structure predictions guided by frameshift sequence-specific and genome-wide experimental data. We build on our previous secondary structure investigation of the singular SARS-CoV-2 68 nt frameshift element sequence, by using Shapify to obtain predictions for 132 extended sequences and including covariation information. Previous investigations have not applied hierarchical folding to extended length SARS-CoV-2 frameshift sequences. By doing so, we simulate the effects of ribosome interaction with the frameshift site, providing insight to biological function. We contribute in-depth discussion to contextualize secondary structure dual-graph motifs for SARS-CoV-2, highlighting the energetic stability of the previously identified 3_8 motif alongside the known dominant 3_3 and 3_6 (native-type) -1 PRF structures. Using a combination of thermodynamic methods and sequence covariation, our novel predictions suggest function of the attenuator hairpin via previously unknown pseudoknotted base pairing. While certain initial RNA folding is consistent, other pseudoknotted base pairs form which indicate potential conformational switching between the two structures.

The myriad roles of RNA structure in the flavivirus life cycle.

As positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

Characterization of nsp1 Binding to the Viral RNA Leader Sequence of Severe Acute Respiratory Syndrome Coronavirus.

Nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus (SCOV1 and SCOV2) acts as a host shutoff protein by blocking the translation of host mRNAs and triggering their decay. Surprisingly, viral RNA, which resembles host mRNAs containing a 5'-cap and a 3'-poly(A) tail, escapes significant translation inhibition and RNA decay, aiding viral propagation. Current literature proposes that, in SCOV2, nsp1 binds the viral RNA leader sequence, and the interaction may serve to distinguish viral RNA from host mRNA. However, a direct binding between SCOV1 nsp1 and the corresponding RNA leader sequence has not been established yet. Here, we show that SCOV1 nsp1 binds to the SCOV1 RNA leader sequence but forms multiple complexes at a high concentration of nsp1. These complexes are marginally different from complexes formed with SCOV2 nsp1. Finally, mutations of the RNA stem-loop did not completely abolish RNA binding by nsp1, suggesting that an RNA secondary structure is more important for binding than the sequence itself. Understanding the nature of binding of nsp1 to viral RNA will allow us to understand how this viral protein selectively suppresses host gene expression.

High frequency of transition to transversion ratio in the stem region of RNA secondary structure of untranslated region of SARS-CoV-2.

Introduction: The propensity of nucleotide bases to form pairs, causes folding and the formation of secondary structure in the RNA. Therefore, purine (R): pyrimidine (Y) base-pairing is vital to maintain uniform lateral dimension in RNA secondary structure. Transversions or base substitutions between R and Y bases, are more detrimental to the stability of RNA secondary structure, than transitions derived from substitutions between A and G or C and T. The study of transversion and transition base substitutions is important to understand evolutionary mechanisms of RNA secondary structure in the 5'  and 3'  untranslated (UTR) regions of SARS-CoV-2. In this work, we carried out comparative analysis of transition and transversion base substitutions in the stem and loop regions of RNA secondary structure of SARS-CoV-2. Methods: We have considered the experimentally determined and well documented stem and loop regions of 5' and 3' UTR regions of SARS-CoV-2 for base substitution analysis. The secondary structure comprising of stem and loop regions were visualized using the RNAfold web server. The GISAID repository was used to extract base sequence alignment of the UTR regions. Python scripts were developed for comparative analysis of transversion and transition frequencies in the stem and the loop regions. Results: The results of base substitution analysis revealed a higher transition (ti) to transversion (tv) ratio (ti/tv) in the stem region of UTR of RNA secondary structure of SARS-CoV-2 reported during the early stage of the pandemic. The higher ti/tv ratio in the stem region suggested the influence of secondary structure in selecting the pattern of base substitutions. This differential pattern of ti/tv values between stem and loop regions was not observed among the Delta and Omicron variants that dominated the later stage of the pandemic. It is noteworthy that the ti/tv values in the stem and loop regions were similar among the later dominant Delta and Omicron variant strains which is to be investigated to understand the rapid evolution and global adaptation of SARS-CoV-2. Conclusion: Our findings implicate the lower frequency of transversions than the transitions in the stem regions of UTRs of SARS-CoV-2. The RNA secondary structures are associated with replication, translation, and packaging, further investigations are needed to understand these base substitutions across different variants of SARS-CoV-2.

Locations and structures of influenza A virus packaging-associated signals and other functional elements via an in silico pipeline for predicting constrained features in RNA viruses.

Influenza A virus contains regions of its segmented genome associated with ability to package the segments into virions, but many such regions are poorly characterised. We provide detailed predictions of the key locations within these packaging-associated regions, and their structures, by applying a recently-improved pipeline for delineating constrained regions in RNA viruses and applying structural prediction algorithms. We find and characterise other known constrained regions within influenza A genomes, including the region associated with the PA-X frameshift, regions associated with alternative splicing, and constraint around the initiation motif for a truncated PB1 protein, PB1-N92, associated with avian viruses. We further predict the presence of constrained regions that have not previously been described. The extra characterisation our work provides allows investigation of these key regions for drug target potential, and points towards determinants of packaging compatibility between segments.

RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies.

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections.

Unveiling hidden structural patterns in the SARS-CoV-2 genome: Computational insights and comparative analysis.

SARS-CoV-2, the causative agent of COVID-19, is known to exhibit secondary structures in its 5' and 3' untranslated regions, along with the frameshifting stimulatory element situated between ORF1a and 1b. To identify additional regions containing conserved structures, we utilized a multiple sequence alignment with related coronaviruses as a starting point. We applied a computational pipeline developed for identifying non-coding RNA elements. Our pipeline employed three different RNA structural prediction approaches. We identified forty genomic regions likely to harbor structures, with ten of them showing three-way consensus substructure predictions among our predictive utilities. We conducted intracomparisons of the predictive utilities within the pipeline and intercomparisons with four previously published SARS-CoV-2 structural datasets. While there was limited agreement on the precise structure, different approaches seemed to converge on regions likely to contain structures in the viral genome. By comparing and combining various computational approaches, we can predict regions most likely to form structures, as well as a probable structure or ensemble of structures. These predictions can be used to guide surveillance, prophylactic measures, or therapeutic efforts. Data and scripts employed in this study may be found at https://doi.org/10.5281/zenodo.8298680.

SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity.

It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However, how nsp15 processes viral dsRNA, commonly considered as a genome replication intermediate, remains elusive. Previous research has mainly focused on short single-stranded RNA as substrates, and whether nsp15 prefers single-stranded or double-stranded RNA for cleavage is controversial. In the present work, we prepared numerous RNA substrates, including both long substrates mimicking the viral genome and short defined RNA, to clarify the substrate preference and cleavage pattern of SARS-CoV-2 nsp15. We demonstrated that SARS-CoV-2 nsp15 preferentially cleaved pyrimidine nucleotides located in less thermodynamically stable areas in dsRNA, such as AU-rich areas and mismatch-containing areas, in a nicking manner. Because coronavirus genomes generally have a high AU content, our work supported the mechanism that coronaviruses evade the antiviral response mediated by host cell dsRNA sensors by using nsp15 dsRNA nickase to directly cleave dsRNA intermediates formed during genome replication and transcription.

Conserved structures and dynamics in 5'-proximal regions of Betacoronavirus RNA genomes.

Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5'-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5'-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.

The application of single-molecule optical tweezers to study disease-related structural dynamics in RNA.

RNA, a dynamic and flexible molecule with intricate three-dimensional structures, has myriad functions in disease development. Traditional methods, such as X-ray crystallography and nuclear magnetic resonance, face limitations in capturing real-time, single-molecule dynamics crucial for understanding RNA function. This review explores the transformative potential of single-molecule force spectroscopy using optical tweezers, showcasing its capability to directly probe time-dependent structural rearrangements of individual RNA molecules. Optical tweezers offer versatility in exploring diverse conditions, with the potential to provide insights into how environmental changes, ligands and RNA-binding proteins impact RNA behaviour. By enabling real-time observations of large-scale structural dynamics, optical tweezers emerge as an invaluable tool for advancing our comprehension of RNA structure and function. Here, we showcase their application in elucidating the dynamics of RNA elements in virology, such as the pseudoknot governing ribosomal frameshifting in SARS-CoV-2.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

The molecular language of RNA 5' ends: guardians of RNA identity and immunity.

RNA caps are deposited at the 5' end of RNA polymerase II transcripts. This modification regulates several steps of gene expression, in addition to marking transcripts as self to enable the innate immune system to distinguish them from uncapped foreign RNAs, including those derived from viruses. Specialized immune sensors, such as RIG-I and IFITs, trigger antiviral responses upon recognition of uncapped cytoplasmic transcripts. Interestingly, uncapped transcripts can also be produced by mammalian hosts. For instance, 5'-triphosphate RNAs are generated by RNA polymerase III transcription, including tRNAs, Alu RNAs, or vault RNAs. These RNAs have emerged as key players of innate immunity, as they can be recognized by the antiviral sensors. Mechanisms that regulate the presence of 5'-triphosphates, such as 5'-end dephosphorylation or RNA editing, prevent immune recognition of endogenous RNAs and excessive inflammation. Here, we provide a comprehensive overview of the complexity of RNA cap structures and 5'-triphosphate RNAs, highlighting their roles in transcript identity, immune surveillance, and disease.

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability.

Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

High-resolution RNA tertiary structures in Zika virus stem-loop A for the development of inhibitory small molecules.

Flaviviruses such as Zika (ZIKV) and dengue virus (DENV) are positive-sense RNA viruses belonging to Flaviviridae The flavivirus genome contains a 5' end stem-loop promoter sequence known as stem-loop A (SLA) that is recognized by the flavivirus polymerase NS5 during viral RNA synthesis and 5' guanosine cap methylation. The crystal structures of ZIKV and DENV SLAs show a well-defined fold, consisting of a bottom stem, side loop, and top stem-loop, providing unique interaction sites for small molecule inhibitors to disrupt the promoter function. To facilitate the identification of small molecule binding sites in flavivirus SLA, we determined high-resolution structures of the bottom and top stems of ZIKV SLA, which contain a single U- or G-bulge, respectively. Both bulge nucleotides exhibit multiple orientations, from folded back on the adjacent nucleotide to flipped out of the helix, and are stabilized by stacking or base triple interactions. These structures suggest that even a single unpaired nucleotide can provide flexibility to RNA structures, and its conformation is mainly determined by the stabilizing chemical environment. To facilitate discovery of small molecule inhibitors that interfere with the functions of ZIKV SLA, we screened and identified compounds that bind to the bottom and top stems of ZIKV SLA.

DinoKnot: Duplex Interaction of Nucleic Acids With PseudoKnots.

Interaction of nucleic acid molecules is essential for their functional roles in the cell and their applications in biotechnology. While simple duplex interactions have been studied before, the problem of efficiently predicting the minimum free energy structure of more complex interactions with possibly pseudoknotted structures remains a challenge. In this work, we introduce a novel and efficient algorithm for prediction of Duplex Interaction of Nucleic acids with pseudoKnots, DinoKnot follows the hierarchical folding hypothesis to predict the secondary structure of two interacting nucleic acid strands (both homo- and hetero-dimers). DinoKnot utilizes the structure of molecules before interaction as a guide to find their duplex structure allowing for possible base pair competitions. To showcase DinoKnots's capabilities we evaluated its predicted structures against (1) experimental results for SARS-CoV-2 genome and nine primer-probe sets, (2) a clinically verified example of a mutation affecting detection, and (3) a known nucleic acid interaction involving a pseudoknot. In addition, we compared our results against our closest competition, RNAcofold, further highlighting DinoKnot's strengths. We believe DinoKnot can be utilized for various applications including screening new variants for potential detection issues and supporting existing applications involving DNA/RNA interactions, adding structural considerations to the interaction to elicit functional information.

Diversity and modularity of tyrosine-accepting tRNA-like structures.

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLSTyr) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5' domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLSTyr compared to those in Bromoviridae Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLSTyr and the 3' domain from a hordeivirus TLSTyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLSTyrs performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLSTyr Finally, these structurally and phylogenetically divergent types of TLSTyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures.