RESUMO
Viruses often pose a significant threat to the host through the exploitation of cellular machineries for their own benefit. In the context of immune responses, myriad host factors are deployed to target viral RNAs and inhibit viral protein translation, ultimately hampering viral replication. Understanding how "non-self" RNAs interact with the host translation machinery and trigger immune responses would help in the development of treatment strategies for viral infections. In this review, we explore how interferon-stimulated gene products interact with viral RNA and the translation machinery in order to induce either global or targeted translation inhibition.
Assuntos
Interferons , Biossíntese de Proteínas , RNA Viral , Viroses , Animais , Humanos , Interações Hospedeiro-Patógeno , Interferons/imunologia , Interferons/metabolismo , Interferons/genética , RNA Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Viroses/imunologia , Viroses/virologia , Viroses/genética , Replicação Viral , Vírus/imunologia , Vírus/genética , Vírus/efeitos dos fármacosRESUMO
Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
Assuntos
COVID-19 , RNA Viral , Humanos , COVID-19/metabolismo , Endonucleases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação ViralRESUMO
Mice are refractory to infection with human-tropic hepatitis C virus (HCV), although distantly related rodent hepaciviruses (RHV) circulate in wild rodents. To investigate whether liver intrinsic host factors can exhibit broad restriction against these distantly related hepaciviruses, we focused on Shiftless (Shfl), an interferon (IFN)-regulated gene (IRG) which restricts HCV in humans. Unusually, and in contrast to selected classical IRGs, human and mouse SHFL orthologues (hSHFL and mSHFL, respectively) were highly expressed in hepatocytes in the absence of viral infection, weakly induced by IFN, and highly conserved at the amino acid level (>95%). Replication of both HCV and RHV subgenomic replicons was suppressed by ectopic expression of mSHFL in human or rodent hepatoma cell lines. Gene editing of endogenous mShfl in mouse liver tumor cells increased HCV replication and virion production. Colocalization of mSHFL protein with viral double-stranded RNA (dsRNA) intermediates was confirmed and could be ablated by mutational disruption of the SHFL zinc finger domain, concomitant with a loss of antiviral activity. In summary, these data point to an evolutionarily conserved function for this gene in humans and rodents: SHFL is an ancient antiviral effector which targets distantly related hepaciviruses via restriction of viral RNA replication. IMPORTANCE Viruses have evolved ways to evade or blunt innate cellular antiviral mechanisms within their cognate host species. However, these adaptations may fail when viruses infect new species and can therefore limit cross-species transmission. This may also prevent development of animal models for human-pathogenic viruses. HCV shows a narrow species tropism likely due to distinct human host factor usage and innate antiviral defenses limiting infection of nonhuman liver cells. Interferon (IFN)-regulated genes (IRGs) partially inhibit HCV infection of human cells by diverse mechanisms. Here, we show that mouse Shiftless (mSHFL), a protein that interferes with HCV replication factories, inhibits HCV replication and infection in human and mouse liver cells. We further report that the zinc finger domain of SHFL is important for viral restriction. These findings implicate mSHFL as a host factor that impairs HCV infection of mice and provide guidance for development of HCV animal models needed for vaccine development.
Assuntos
Hepacivirus , Hepatite C , Camundongos , Humanos , Animais , Hepacivirus/genética , Antivirais/farmacologia , Interferons , Fatores de Restrição AntiviraisRESUMO
The RNA genome of SARS-CoV-2 contains a frameshift stimulatory element (FSE) that allows access to an alternative reading frame through -1 programmed ribosomal frameshifting (PRF). -1PRF in the 1a/1b gene is essential for efficient viral replication and transcription of the viral genome. -1PRF efficiency relies on the presence of conserved RNA elements within the FSE. One of these elements is a three-stemmed pseudoknot, although alternative folds of the frameshift site might have functional roles as well. Here, by complementing ensemble and single-molecule structural analysis of SARS-CoV-2 frameshift RNA variants with functional data, we reveal a conformational interplay of the 5' and 3' immediate regions with the FSE and show that the extended FSE exists in multiple conformations. Furthermore, limiting the base pairing of the FSE with neighboring nucleotides can favor or impair the formation of the alternative folds, including the pseudoknot. Our results demonstrate that co-existing RNA structures can function together to fine-tune SARS-CoV-2 gene expression, which will aid efforts to design specific inhibitors of viral frameshifting.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , SARS-CoV-2 , Humanos , COVID-19 , Mudança da Fase de Leitura do Gene Ribossômico/genética , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/química , SARS-CoV-2/genética , SARS-CoV-2/fisiologiaRESUMO
Optical tweezers are a single-molecule technique that allows probing of intra- and intermolecular interactions that govern complex biological processes involving molecular motors, protein-nucleic acid interactions, and protein/RNA folding. Recent developments in instrumentation eased and accelerated optical tweezers data acquisition, but analysis of the data remains challenging. Here, to enable high-throughput data analysis, we developed an automated python-based analysis pipeline called POTATO (practical optical tweezers analysis tool). POTATO automatically processes the high-frequency raw data generated by force-ramp experiments and identifies (un)folding events using predefined parameters. After segmentation of the force-distance trajectories at the identified (un)folding events, sections of the curve can be fitted independently to a worm-like chain and freely jointed chain models, and the work applied on the molecule can be calculated by numerical integration. Furthermore, the tool allows plotting of constant force data and fitting of the Gaussian distance distribution over time. All these features are wrapped in a user-friendly graphical interface, which allows researchers without programming knowledge to perform sophisticated data analysis.
Assuntos
Pinças Ópticas , Solanum tuberosum , Nanotecnologia/métodos , Dobramento de Proteína , RNARESUMO
RNA dimerization is the noncovalent association of two human immunodeficiency virus-1 (HIV-1) genomes. It is a conserved step in the HIV-1 life cycle and assumed to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. Here, we developed functional analysis of RNA structure-sequencing (FARS-seq) to comprehensively identify sequences and structures within the HIV-1 5' untranslated region (UTR) that regulate this critical step. Using FARS-seq, we found nucleotides important for dimerization throughout the HIV-1 5' UTR and identified distinct structural conformations in monomeric and dimeric RNA. In the dimeric RNA, key functional domains, such as stem-loop 1 (SL1), polyadenylation signal (polyA) and primer binding site (PBS), folded into independent structural motifs. In the monomeric RNA, SL1 was reconfigured into long- and short-range base pairings with polyA and PBS, respectively. We show that these interactions disrupt genome packaging, and additionally show that the PBS-SL1 interaction unexpectedly couples the PBS with dimerization and Pr55Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.
Assuntos
HIV-1 , Regiões 5' não Traduzidas/genética , Dimerização , HIV-1/fisiologia , Humanos , Conformação de Ácido Nucleico , RNA Viral/química , Proteínas Virais/metabolismoRESUMO
CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defence against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes transactivating crRNA (tracrRNA) hybridization with the second of the two repeats, accelerating crRNA processing. Accordingly, disruption of this structure reduces the abundance of the associated crRNA and immune defence against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defence against the most recently encountered invaders.
Assuntos
Bacteriófagos , Proteínas Associadas a CRISPR , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , RNA/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismoRESUMO
Translation facilitates the transfer of the genetic information stored in the genome via messenger RNAs to a functional protein and is therefore one of the most fundamental cellular processes. Programmed ribosomal frameshifting is a ubiquitous alternative translation event that is extensively used by viruses to regulate gene expression from overlapping open reading frames in a controlled manner. Recent technical advances in the translation field enabled the identification of precise mechanisms as to how and when ribosomes change the reading frame on mRNAs containing cis-acting signals. Several studies began also to illustrate that trans-acting RNA modulators can adjust the timing and efficiency of frameshifting illuminating that frameshifting can be a dynamically regulated process in cells. Here, we intend to summarize these new findings and emphasize how it fits in our current understanding of PRF mechanisms as previously described.
RESUMO
RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously. This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.
Assuntos
COVID-19 , Pinças Ópticas , Humanos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/genética , SARS-CoV-2RESUMO
Cardioviruses are single-stranded RNA viruses of the family Picornaviridae. In addition to being the first example of internal ribosome entry site (IRES) utilization, cardioviruses also employ a series of alternative translation strategies, such as Stop-Go translation and programmed ribosome frameshifting. Here, we focus on cardiovirus 2A protein, which is not only a primary virulence factor, but also exerts crucial regulatory functions during translation, including activation of viral ribosome frameshifting and inhibition of host cap-dependent translation. Only recently, biochemical and structural studies have allowed us to close the gaps in our knowledge of how cardiovirus 2A is able to act in diverse translation-related processes as a novel RNA-binding protein. This review will summarize these findings, which ultimately may lead to the discovery of other RNA-mediated gene expression strategies across a broad range of RNA viruses.
Assuntos
Cardiovirus/genética , Sítios Internos de Entrada Ribossomal , Proteínas Virais , Sítios Internos de Entrada Ribossomal/genética , Proteínas Virais/genéticaRESUMO
Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
Assuntos
Infecções por Cardiovirus/virologia , Vírus da Encefalomiocardite/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Virais/química , Proteínas Virais/metabolismo , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/metabolismo , Cristalografia por Raios X , Vírus da Encefalomiocardite/química , Vírus da Encefalomiocardite/genética , Mudança da Fase de Leitura do Gene Ribossômico , Humanos , Ribossomos/genética , Ribossomos/metabolismo , Proteínas Virais/genéticaRESUMO
Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , SARS-CoV-2/genética , COVID-19 , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Conformação de Ácido Nucleico , Isoformas de Proteínas , Proteoma , RNA Viral/genética , SARS-CoV-2/fisiologia , Replicação ViralRESUMO
The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Proteínas Virais/metabolismo , Ribossomos/metabolismo , Theilovirus/genética , Theilovirus/metabolismo , Proteínas Virais/químicaRESUMO
Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.
Assuntos
COVID-19/metabolismo , COVID-19/virologia , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo , Autoantígenos/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Mapas de Interação de Proteínas , Proteoma , RNA Viral/genética , Ribonucleoproteínas/metabolismo , SARS-CoV-2/genética , Replicação Viral/fisiologia , Antígeno SS-BRESUMO
mRNA contexts containing a 'slippery' sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the -1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how -1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and -1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA-mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.
Assuntos
Proteínas de Bactérias/genética , DNA Polimerase III/genética , Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Modelos Teóricos , Pareamento de Bases , Teorema de Bayes , Códon , Mutação da Fase de Leitura , Lisina/genética , Fenilalanina/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , TermodinâmicaRESUMO
Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Regulação da Expressão Gênica/efeitos dos fármacos , RNA/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sequência de Bases/genética , Carbamatos/síntese química , Carbamatos/química , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Naftiridinas/síntese química , Naftiridinas/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Isoformas de Proteínas/genética , RNA/química , RNA Mensageiro/genética , RNA Viral/genética , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Biologia SintéticaRESUMO
Ribosome frameshifting during translation of bacterial dnaX can proceed via different routes, generating a variety of distinct polypeptides. Using kinetic experiments, we show that -1 frameshifting predominantly occurs during translocation of two tRNAs bound to the slippery sequence codons. This pathway depends on a stem-loop mRNA structure downstream of the slippery sequence and operates when aminoacyl-tRNAs are abundant. However, when aminoacyl-tRNAs are in short supply, the ribosome switches to an alternative frameshifting pathway that is independent of a stem-loop. Ribosome stalling at a vacant 0-frame A-site codon results in slippage of the P-site peptidyl-tRNA, allowing for -1-frame decoding. When the -1-frame aminoacyl-tRNA is lacking, the ribosomes switch into -2 frame. Quantitative mass spectrometry shows that the -2-frame product is synthesized in vivo. We suggest that switching between frameshifting routes may enrich gene expression at conditions of aminoacyl-tRNA limitation.
Assuntos
Proteínas de Bactérias/biossíntese , DNA Polimerase III/biossíntese , Escherichia coli/enzimologia , Mudança da Fase de Leitura do Gene Ribossômico , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Bactérias/genética , DNA Polimerase III/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Cinética , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Espectrometria de Massas em TandemRESUMO
During translation elongation, ribosome translocation along an mRNA entails rotations of the ribosomal subunits, swiveling motions of the small subunit (SSU) head and stepwise movements of the tRNAs together with the mRNA. Here, we reconstructed the choreography of the collective motions of the Escherichia coli ribosome during translocation promoted by elongation factor EF-G, by recording the fluorescence signatures of nine different reporters placed on both ribosomal subunits, tRNA and mRNA. We captured an early forward swiveling of the SSU head taking place while the SSU body rotates in the opposite, clockwise direction. Backward swiveling of the SSU head starts upon tRNA translocation and continues until the post-translocation state is reached. This work places structures of translocation intermediates along a time axis and unravels principles of the motions of macromolecular machines.