Biology subjects

Kieft, J. S.

Publications and source records attributed to Kieft, J. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Enterovirus RNase L inhibiting RNAs are highly conserved with limited phylogenetic distribution

RNA molecules form specific 3D structures that facilitate a variety of functions through interactions with other macromolecules. Many RNA viral genomes maintain these structures to interact with and evade host immunity machinery. One such element, the competitive inhibitor RNA (ciRNA), discovered in the protein coding region of the poliovirus serotype 1 (PV1) genome, inhibits a host antiviral protein, ribonuclease L (RNase L). Although some functionally essential structural motifs of the PV1 ciRNA have been studied, the extent of its evolutionary conservation and other structural requirements remained unexplored. Here we combined bioinformatic and biochemical techniques to further define the requirements of a functional ciRNA and assess its phylogenetic distribution. We systematically mutated ciRNA structural features, verifying that ciRNA inhibitory activity requires a conserved loop E motif and a long-range base-pairing interaction, but its peripheral stems are dispensable and in fact a circularly permuted version maintains function. A structure-based homology search identified potential ciRNAs across the Picornaviridae family, but only a subset of those tested were functional--all are in Enterovirus coxsackiepol. When structural features needed for function were transposed from PV1 ciRNA to an RNA unable to inhibit RNase L, the chimeric RNAs did not gain wild-type function, and chemical probing data revealed that these nonfunctional RNAs are unable to form the correct secondary structure. Overall, the dual constraints of encoding a protein and forming a specific functional structure appear to not only limit the sequence diversity, but also the phylogenetic distribution, of ciRNAs.

microbiology↗

bioRxiv2026

An A-rich linker between dengue virus tandem xrRNAs facilitates functional coordination

Orthoflaviviruses use programmed resistance to host 5' to 3' exoribonucleases to produce subgenomic flaviviral RNAs (sfRNAs) during infection. This resistance is conferred by exoribonuclease-resistant RNA (xrRNA) structures that often occur in tandem and whose function can be coupled. In dengue virus serotype 2 (DENV2) this coupling results in changing patterns of sfRNA identity and abundance linked to the ability of the virus to adapt to host vs. vector infections. The physical basis of this coupling was unknown. Using a combination of virology, biochemistry, bioinformatics, structural biology, and biophysics, we explored the structural and sequence determinants of tandem xrRNA coupling in DENV2. We discovered that the spatial proximity, order, and structural integrity of the tandem xrRNAs are all important for coupling. Furthermore, an unpaired A-rich linker that lies between the two xrRNAs is essential in stabilizing a specific structure that correlates to coupling. This A-rich sequence likely forms tertiary contacts with an adjacent stem-loop structure to form a physical bridge between the two xrRNAs, a finding that is supported by a mid-resolution cryoEM map of the DENV2 tandem xrRNAs. Disruption of the structure of this bridge by mutation changes the relative orientation or spacing between the tandem xrRNAs, which is correlated to their functional coupling. These findings provide an explanation for the coupling between tandem xrRNAs and suggests new mechanistic hypotheses. IMPORTANCEDengue virus (DENV) generates non-coding subgenomic flaviviral RNAs (sfRNAs) that affect several cellular pathways and are important for successful infection. These sfRNAs are formed by structured RNA elements in the viral genome called exoribonuclease-resistant RNAs (xrRNAs), which fold into a distinct three-dimensional topology to block degradation by host cell exoribonucleases and often occur in tandem. Specific patterns of sfRNAs made during infection are important for host vs. vector fitness, and in DENV2 this pattern depends on functional coupling between tandem xrRNAs. However, the source of this functional coupling was unknown. We determined that an unpaired A-rich linker between the tandem xrRNAs is necessary for creating a structural bridge between the tandem xrRNAs. This bridge appears to favor a specific orientation between the tandem xrRNAs that is correlated to coupling and therefore to the patterns and relative abundance of sfRNAs produced during infection.

microbiology↗

bioRxiv2025

A compact viral IRES translates a downstream open reading frame

Hepatitis C virus (HCV) and many other RNA viruses contain a Type IV internal ribosome entry site (IRES) in their 5' untranslated region (UTR). These IRES RNAs adopt a complex tertiary structure that interacts directly with the ribosome, enabling cap-independent translation initiation. Using bioinformatic methods to search viral genomes for more Type IV IRES RNAs, we discovered that megrivirus E (MeV-E) contains a putative Type IV IRES within its annotated 3' UTR. In addition to its unusual location downstream of the main coding region, the size of the MeV-E 3' IRES is substantially reduced compared to known Type IV IRESs. We confirmed the secondary structure of the MeV-E 3' IRES, determined its 3D structure in complex with the ribosome using cryoEM, and showed that the MeV-E 3' IRES initiates translation but at lower levels compared to the larger Type IV IRES in the MeV-E 5' UTR. We hypothesize that the absence of several domains in the MeV-E 3' IRES compared to other Type IV IRESs results in a loss of ribosomal protein interactions and a relative decrease in translation activity. This small Type IV IRES enables translation of a second open reading frame in the MeV-E genome, which likely encodes a transmembrane protein that is conserved in other megriviruses. We propose a model wherein MeV-E expresses lower levels of its downstream-encoded protein compared to those in the upstream coding region using a pared down IRES structure, demonstrating purposeful tuning of translation through RNA structural variation.

microbiology↗

bioRxiv2025

Distribution and structural diversity of Type IV internal ribosome entry sites

Internal Ribosome Entry Sites (IRESs) are RNAs that facilitate cap- and end-independent translation initiation in eukaryotes. Type IV IRESs, which include the hepatitis C virus IRES, directly bind the 40S subunit and require only a subset of the canonical initiation factors to function. As the full diversity and species distribution of these IRESs was unknown, we sought to identify and classify their full architectural variation. Using a secondary structure homology-based search method, we identified 163 putative Type IV IRESs from viruses with diverse hosts and phylogeny, including the first example in a double stranded viral genome. Clustering analysis based on the presence and overall size of secondary structure elements yielded three distinct groups, differentiated by secondary structure expansions and deletions. Chemical probing of representative IRESs from each cluster validated the predicted secondary structures and in vitro translation assays showed that structural differences correlate with functional variation. Our findings reveal distinct structural adaptations and patterns within the Type IV IRESs that may influence IRES function and mechanism.

microbiology↗

bioRxiv2024

Tick-borne flavivirus exoribonuclease-resistant RNAs contain a 'double loop' structure

Flaviviridae viruses are human pathogens that generate subgenomic noncoding RNAs during infection using structured exoribonuclease resistant RNAs (xrRNAs) that block progression of host cells exoribonucleases. The structures of several xrRNAs from mosquito-borne and insect-specific flaviviruses have been solved, revealing a conserved fold in which a ring-like motif encircles the end of the xrRNA. However, the xrRNAs found in tick-borne and no known vector flaviviruses have distinct characteristics and their 3-D fold was unsolved. To address this, we identified subgenomic flaviviral RNA formation in the encephalitis-causing tick-borne Powassan Virus. We characterized their secondary structure using chemical probing and solved the structure of one of its xrRNAs using cryo-EM. This structure reveals a novel double loop ring element leading to a model in which the ring is remodeled upon encountering the exoribonuclease. Using bioinformatic analyses we showed that this structure is representative of a broad class of xrRNAs and defined key structural and sequence determinants of function. These discoveries reveal a conserved strategy of structure-based exoribonuclease resistance achieved through a unique topology across a viral family of key importance to global health.

microbiology↗

bioRxiv2023

A conserved class of viral RNA structures regulate translation reinitiation through dynamic ribosome interactions

Certain viral RNAs encode proteins downstream of the main protein coding region, expressed through "termination-reinitiation" events, dependent on RNA structure. RNA elements located upstream of the first stop codon within these viral mRNAs bind the ribosome, preventing ribosome recycling and inducing reinitiation. We used bioinformatic methods to identify new examples of viral reinitiation-stimulating RNAs and experimentally verified their secondary structure and function. We determined the structure of a representative viral RNA-ribosome complex using cryoEM. 3D classification and variability analyses reveal that the viral RNA structure can sample a range of conformations while remaining tethered to the ribosome, which enabling the ribosome to find a reinitiation start site within a limited range of mRNA sequence. Evaluating the conserved features and constraints of this entire RNA class in the context of the cryoEM reconstruction provides insight into mechanisms enabling reinitiation, a translation regulation strategy employed by many other viral and eukaryotic systems.

molecular biology↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2023

A Generalizable Scaffold-Based Approach for Structure Determination of RNAs by Cryo-EM

Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules remain challenging targets due to their intrinsic low signal to noise ratio. Methods to resolve small proteins have been applied but development of similar approaches for small structured RNA elements have lagged. Here, we present a scaffold-based approach that we used to recover maps of sub-25 kDa RNA domains to 4.5 - 5.0 [A]. While lacking the detail of true high-resolution maps, these are suitable for model building and preliminary structure determination. We demonstrate this method faithfully recovers the structure of several RNA elements of known structure, and it promises to be generalized to other RNAs without disturbing their native fold. This approach may streamline the sample preparation process and reduce the optimization required for data collection. This first-generation scaffold approach provides a system for RNA structure determination by cryo-EM and lays the groundwork for further scaffold optimization to achieve higher resolution.

biophysics↗

bioRxiv2023

An evolutionarily conserved strategy for ribosome binding and inhibition by β-coronavirus non-structural protein 1

An important pathogenicity factor of SARS-CoV-2 and related coronaviruses is Nsp1, which suppresses host gene expression and stunts antiviral signaling. SARS-CoV-2 Nsp1 binds the ribosome to inhibit translation through mRNA displacement and induces degradation of host mRNAs through an unknown mechanism. Here we show that Nsp1-dependent host shutoff is conserved in diverse coronaviruses, but only Nsp1 from {beta}-CoV inhibits translation through ribosome binding. The C-terminal domain of all {beta}-CoV Nsp1s confers high-affinity ribosome-binding despite low sequence conservation. Modeling of interactions of four Nsp1s to the ribosome identified few absolutely conserved amino acids that, together with an overall conservation in surface charge, form the {beta}-CoV Nsp1 ribosome-binding domain. Contrary to previous models, the Nsp1 ribosome-binding domain is an inefficient translation inhibitor. Instead, the Nsp1-CTD likely functions by recruiting Nsp1s N-terminal "effector" domain. Finally, we show that a viral cis-acting RNA element has co-evolved to fine-tune SARS-CoV-2 Nsp1 function, but does not provide similar protection against Nsp1 from related viruses. Together, our work provides new insight into the diversity and conservation of ribosome-dependent host-shutoff functions of Nsp1, knowledge that could aide future efforts in pharmacological targeting of Nsp1 from SARS-CoV-2, but also related human-pathogenic {beta}-coronaviruses. Our study also exemplifies how comparing highly divergent Nsp1 variants can help to dissect the different modalities of this multi-functional viral protein.

biochemistry↗

bioRxiv2023

Zika virus dumbbell-1 structure is critical for sfRNA presence and cytopathic effect during infection

Zika virus (ZIKV) contains multiple conserved RNA structures in the viral 3 untranslated region (UTR), including the structure known as dumbbell-1 (DB-1). Previous research has shown that the DB-1 structure is important for flavivirus genome replication and cytopathic effect (CPE). However, the role of the DB structure and the mechanism by which it contributes to viral pathogenesis is not known. Using recently solved flavivirus DB RNA structural data, we designed two DB-1 mutant ZIKV infectious clones termed ZIKV-TL.PK, which disrupts DB-1 tertiary folding and ZIKV-p.2.5, which alters DB-1 secondary structure formation. In cell culture, we found that viral genome replication of both mutant clones is not significantly affected compared to ZIKV-WT, but viral CPE is considerably decreased. We investigated sub-genomic flaviviral RNA (sfRNA) formation by both DB-1 mutants following A549 infection and found both mutant clones have decreased levels of all sfRNA species compared to ZIKV-WT during infection. To investigate the mechanism of decreased CPE in our DB-1 mutant clones, we assayed ZIKV DB mutant-infected A549 cells for cell viability and caspase activation. We found that cell viability is significantly increased in DB-1 mutant-infected cells compared to ZIKV-WT due to reduced caspase 3 activation. We also show that replication of the ZIKV-P.2.5 mutant was significantly restricted by type I interferon treatment without altering interferon stimulated gene expression. Using a murine model of ZIKV infection, we show that both ZIKV-DB-1 mutants exhibit reduced morbidity and mortality compared to ZIKV-WT virus due to tissue specific attenuation in ZIKV-DB viral replication in the brain tissue. Overall, our data show that the flavivirus DB-1 RNA structure is important for maintaining sfRNA levels during infection which supports caspase-3 dependent, viral cytopathic effect, type 1 interferon resistance, and viral pathogenesis in a mouse model.

microbiology↗

bioRxiv2022

Improved methods for the large-scale cultivation of Giardia lamblia trophozoites in the academic laboratory

The parasitic protist Giardia lamblia causes giardiasis, one of the leading diarrheal diseases worldwide. As a phylogenetically divergent eukaryote, Giardia is also an emerging model organism for exploring the diversity of core eukaryotic metabolic pathways and important molecular structures. To date, methods for the cultivation of large numbers (>109) of Giardia trophozoites have been either expensive or cumbersome, hampering structural and biochemical studies. Here, we present two improved strategies for the laboratory-scale growth of billions of adherent trophozoites, with suggestions for the optimization of media components, media preparation, and harvesting methods.

microbiology↗

bioRxiv2022

Cryo-EM reveals an entangled kinetic trap in the folding pathway of a catalytic RNA

Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of such misfolded states, but they are difficult to characterize due to their transient and potentially conformationally dynamic nature. We used cryo-electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the Tetrahymena thermophila group I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally, as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.

biochemistry↗

bioRxiv2022

Therapeutic resistance to FLT3 inhibition is driven by a novel ATM/mTOR pathway regulating oxidative phosphorylation

While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance towards improving therapeutic outcomes. In the present study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn up-regulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance, and a promising strategy for improved treatment of FLT3 mutant AML patients.

cancer biology↗

bioRxiv2020

An Expanded Class of Histidine-Accepting Viral tRNA-like Structures

Structured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLSHis) are examples found at the 3' termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate several processes important for infection, to include replication. As only five TLSHis examples had been reported, we explored the possible larger phylogenetic distribution and diversity of this TLS class using bioinformatic approaches. We identified many new examples of TLSHis, yielding a rigorous consensus sequence and secondary structure model that we validated by chemical probing of representative TLSHis RNAs. We confirmed new examples as authentic TLSHis by demonstrating their ability to be histidylated in vitro, then used mutational analyses to verify a tertiary interaction that is likely analogous to the D- and T-loop interaction found in canonical tRNAs. These results expand our understanding of how diverse RNA sequences achieve tRNA-like structures and functions in the context of viral RNA genomes and lay the groundwork for high-resolution structural studies of tRNA mimicry by histidine-accepting TLSs.

biochemistry↗

bioRxiv2020

The Giardia lamblia ribosome structure reveals divergence in translation and quality control pathways

Giardia lamblia is a human pathogen of worldwide importance with limited treatment options. Its unusual molecular biology presents targets for new therapies and the opportunity to explore the fundamental features of important biological mechanisms. We determined the structure of the G. lamblia 80S ribosome by cryoelectron microscopy, revealing how it combines eukaryotic and bacterial features. The structure reveals regions that are rapidly evolving, including depletion of A and U bases from its rRNA. Specific features of the G. lamblia ribosome suggest it is less prone to stall on problematic peptide sequences, and that the organism uses altered ribosome quality control pathways compared to other eukaryotes. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. This work defines key new questions regarding ribosome-centric biological pathways in G. lamblia and motivates new experiments to explore potential targetable mechanisms.

molecular biology↗

bioRxiv2020

A structured viral RNA uses molecular mimicry and conformational dynamics to coordinate multiple functions

Viruses require multifunctional structured RNAs to hijack their hosts biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo-EM, we visualized the structure of the mysterious viral tRNA-like structure (TLS) from brome mosaic virus (BMV), which affects replication, translation, and genome encapsidation. Structures in isolation and bound to tyrosyl-tRNA synthetase (TyrRS) show that this [~]55 kDa purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs dramatically from tRNA. Our studies reveal how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions and highlights the utility of cryo-EM for visualizing small conformationally dynamic structured RNAs.

biophysics↗

bioRxiv2020

Three-dimensional structure of a flavivirus dumbbell RNA reveals molecular details of an RNA regulator of replication

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5' and 3' untranslated regions (UTRs). The 3' UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs) that regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we used x-ray crystallography and structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.

microbiology↗

bioRxiv2020

Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA for destruction. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific 3-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana Bat Virus (TABV) and solved its structure by x-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

biochemistry↗