Search Results (27)

Members of the Picornaviridae family of viruses have single-stranded positive-sense RNA genomes, and are conventionally considered to contain a single open reading frame (ORF) preceded by a long 5′ untranslated region (UTR). The 5′UTR contains an internal ribosomal entry site (IRES) that mediates end-independent initiation of translation of ORF1, resulting in synthesis of a polyprotein that is proteolytically processed to yield the individual structural and nonstructural proteins. However, recent studies indicate that the dogma that picornavirus genomes contain a single ORF (ORF1) is an oversimplification, and that members of many picornavirus genera contain an additional ORF that is translated to yield an accessory protein that may influence pathogenicity and tissue specificity. This review summarizes the structural organization of picornavirus genomes that contain a second ORF. Most commonly, these ORFs overlap the 5′-end of ORF1, but they may also be wholly distinct from ORF1 or located entirely within it in a different reading frame. Ribosomal access to these alternate ORFs has been shown or is hypothesized to depend on a remarkable variety of non-canonical translation mechanisms, including ribosomal recruitment by a second IRES, leaky scanning after IRES-mediated internal ribosomal entry, cap-independent initiation, frame-shifting and termination–reinitiation. Full article

Antisense oligonucleotides (ASOs) are nucleic acid therapeutics that regulate gene expression through sequence-specific hybridization with target RNA. Under physiological conditions, many target RNAs adopt higher-order structures, which can strongly influence ASO accessibility and binding behavior. Although UV melting analysis is widely used to evaluate the thermal stability of ASO/RNA duplexes, this approach does not adequately account for the structural features of target RNAs. In this study, we investigated the utility of surface plasmon resonance (SPR) analysis as an in vitro method to evaluate ASO binding while considering RNA structural constraints. Multiple ASOs were designed to target PRF84, an 84-nucleotide RNA motif that induces −1 programmed ribosomal frameshifting in HIV-1 gag-pol expression. SPR analyses were performed to compare ASO interactions with complementary RNA fragments and with structurally folded PRF84. The results demonstrated that identical ASOs exhibited distinct binding behaviors depending on whether the target was a complementary RNA or PRF84, indicating that RNA structure significantly affects ASO binding. These findings suggest that SPR analysis enables the evaluation of ASO–RNA interactions taking structure into account, and may be a useful alternative approach to conventional UV melting analysis-based ASO screening. Full article

Bacteriophages are powerful drivers of microbial evolution and are increasingly explored as alternatives to antibiotics against multidrug-resistant pathogens such as Pseudomonas aeruginosa. Here, we describe the isolation, phenotypic characterization and genomic, structural and evolutionary analysis of Pseudomonas phage Adele, a lytic myovirus representing a novel species within the genus Pakpunavirus (family Vandenendeviridae). Phage Adele exhibits a short latent period of 20 min, a burst size of 59 ± 11 virions per infected cell and a high virulence index, efficiently lysing non-O11 Pseudomonas aeruginosa strains and reducing biofilm biomass. In vivo, Adele confers marked protection in a Galleria mellonella infection model. Phylogenetic reconstruction, synteny analysis and structural modeling demonstrate the relatedness of Vandenendeviridae to phages of the Andersonviridae and Vequintavirinae clades, pointing to a stable, ancestral virion architecture that has undergone lineage-specific elaborations, including the duplication and divergence of tail tube proteins. The tail assembly chaperone gene employs a conserved −1 programmed ribosomal frameshift. Phage Adele encodes an elaborate set of metabolic reprogramming and anti-defense systems, reflecting extensive horizontal gene transfer. The combination of a conserved structural architecture and mosaic genome establishes Adele as an exemplary system for studying modular evolution in phages, alongside its demonstrated therapeutic efficacy. Full article

Human immunodeficiency virus (HIV) continues to be a threat to public health. An emerging technique with promise in the context of fighting HIV type 1 (HIV-1) focuses on targeting ribosomal frameshifting. A crucial –1 programmed ribosomal frameshift (PRF) has been observed in several pathogenic viruses, including HIV-1. Altered folds of the HIV-1 RNA frameshift element (FSE) have been shown to alter frameshifting efficiency. Here, we use RNA-As-Graphs (RAG), a graph-theory based framework for representing and analyzing RNA secondary structures, to perform conformational analysis in motif space to propose how sequence length may influence folding patterns. This combined analysis, along with all-atom modeling and experimental testing of our designed mutants, has already proven valuable for the SARS-CoV-2 FSE. As a first step to launching the same computational/experimental approach for HIV-1, we compare prior experiments and perform SHAPE-guided 2D-fold predictions for the HIV-1 FSE embedded in increasing sequence contexts and predict structure-altering mutations. We find a highly stable upper stem and highly flexible lower stem for the core FSE, with a three-way junction connecting to other motifs at increasing lengths. In particular, we find little support for a pseudoknot or triplex interaction in the core FSE, although pseudoknots can form separately as a connective motif at longer sequences. We also identify sensitive residues in the upper stem and central loop that, when minimally mutated, alter the core stem loop folding. These insights into the FSE fold and structure-altering mutations can be further pursued by all-atom simulations and experimental testing to advance the mechanistic understanding and therapeutic strategies for HIV-1. Full article

Programmed Ribosomal Frameshifting (PRF) is a mechanism that alters the mRNA reading frame during translation, resulting in the production of out-of-frame proteins. PRF plays crucial roles in maintaining cellular homeostasis and contributes significantly to disease pathogenesis, particularly in viral infections. Notably, PRF can induce immune responses in the SARS-CoV-2 mRNA vaccine, further extending its biological significance. These multiple aspects of PRF highlight its potential as a therapeutic target. Since PRF efficiency can be modulated by cellular factors, its expression or silencing is context-dependent. Therefore, a deeper understanding of PRF is essential for harnessing its therapeutic potential. This review explores PRF biological significance in disease and homeostasis. Such knowledge would serve as a foundation to advance therapeutic strategies targeting PRF modulation, especially in viral infections and vaccine development. Full article

Sequence analysis of the Zaire ebolavirus (EBOV) polymerase (L gene) mRNA, using online tools, identified a highly ranked −1 programmed ribosomal frameshift (FS) signal including an ideal slippery sequence heptamer (UUUAAAA), with an overlapping coding region featuring two tandem UGA codons, immediately followed by an RNA region that is the inverse complement (antisense) to a region of the mRNA of the selenoprotein iodothyronine deiodinase II (DIO2). This antisense interaction was confirmed in vitro via electrophoretic gel shift assay, using cDNAs at the EBOV and DIO2 segments. The formation of a duplex between the two mRNAs could trigger the ribosomal frameshift, by mimicking the enhancing role of a pseudoknot structure, while providing access to the selenocysteine insertion sequence (SECIS) element contained in the DIO2 mRNA. This process would allow the −1 frame UGA codons to be recoded as selenocysteine, forming part of a C-terminal module in a low abundance truncated isoform of the viral polymerase, potentially functioning in a redox role. Remarkably, 90 bases downstream of the −1 FS site, an active +1 FS site can be demonstrated, which, via a return to the zero frame, would enable the attachment of the entire C-terminal of the polymerase protein. Using a construct with upstream and downstream reporter genes, spanning a wildtype or mutated viral insert, we show significant +1 ribosomal frameshifting at this site. Acting singly or together, frameshifting at these sites (both of which are highly conserved in EBOV strains) could enable the expression of several modified isoforms of the polymerase. The 3D modeling of the predicted EBOV polymerase FS variants using the AI tool, AlphaFold, reveals a peroxiredoxin-like active site with arginine and threonine residues adjacent to a putative UGA-encoded selenocysteine, located on the back of the polymerase “hand”. This module could serve to protect the viral RNA from peroxidative damage. Full article

Type I interferons (IFN-Is) are pivotal in innate immunity against human immunodeficiency virus I (HIV-1) by eliciting the expression of IFN-stimulated genes (ISGs), which encompass potent host restriction factors. While ISGs restrict the viral replication within the host cell by targeting various stages of the viral life cycle, the lesser-known IFN-repressed genes (IRepGs), including RNA-binding proteins (RBPs), affect the viral replication by altering the expression of the host dependency factors that are essential for efficient HIV-1 gene expression. Both the host restriction and dependency factors determine the viral replication efficiency; however, the understanding of the IRepGs implicated in HIV-1 infection remains greatly limited at present. This review provides a comprehensive overview of the current understanding regarding the impact of the RNA-binding protein families, specifically the two families of splicing-associated proteins SRSF and hnRNP, on HIV-1 gene expression and viral replication. Since the recent findings show specifically that SRSF1 and hnRNP A0 are regulated by IFN-I in various cell lines and primary cells, including intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs), we particularly discuss their role in the context of the innate immunity affecting HIV-1 replication. Full article

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis. Full article

The interferon-induced host cell protein Shiftless (SFL) inhibits −1 programmed ribosomal frameshifting (−1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits −1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of −1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as −1PRF signals, both of which are regulated by mRNA secondary structure elements. Full article

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on −1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) is globally prevalent and seriously harms the economic efficiency of pig farming. Because of its immunosuppression and high incidence of mutant recombination, PRRSV poses a great challenge for disease prevention and control. Nonstructural protein 2 (NSP2) is the most variable functional protein in the PRRSV genome and can generate NSP2N and NSP2TF variants due to programmed ribosomal frameshifts. These variants are broad and complex in function and play key roles in numerous aspects of viral protein maturation, viral particle assembly, regulation of immunity, autophagy, apoptosis, cell cycle and cell morphology. In this paper, we review the structural composition, programmed ribosomal frameshift and biological properties of NSP2 to facilitate basic research on PRRSV and to provide theoretical support for disease prevention and control and therapeutic drug development. Full article

A longstanding paradox in molecular biology has centered on the question of how very long proteins are synthesized, despite numerous measurements indicating that ribosomes spontaneously shift reading frame at rates that should preclude their ability completely translate their mRNAs. Shiftless (SFL; C19orf66) was originally identified as an interferon responsive gene encoding an antiviral protein, indicating that it is part of the innate immune response. This activity is due to its ability to bind ribosomes that have been programmed by viral sequence elements to shift reading frame. Curiously, Shiftless is constitutively expressed at low levels in mammalian cells. This study examines the effects of altering Shiftless homeostasis, revealing how it may be used by higher eukaryotes to identify and remove spontaneously frameshifted ribosomes, resolving the apparent limitation on protein length. Data also indicate that Shiftless plays a novel role in the ribosome-associated quality control program. A model is proposed wherein SFL recognizes and arrests frameshifted ribosomes, and depending on SFL protein concentrations, either leads to removal of frameshifted ribosomes while leaving mRNAs intact, or to mRNA degradation. We propose that SFL be added to the growing pantheon of proteins involved in surveilling translational fidelity and controlling gene expression in higher eukaryotes. Full article

The majority of antivirals available target viral proteins; however, RNA is emerging as a new and promising antiviral target due to the presence of highly structured RNA in viral genomes fundamental for their replication cycle. Here, we discuss methods for the identification of RNA-targeting compounds, starting from the determination of RNA structures either from purified RNA or in living cells, followed by in silico screening on RNA and phenotypic assays to evaluate viral inhibition. Moreover, we review the small molecules known to target the programmed ribosomal frameshifting element of SARS-CoV-2, the internal ribosomal entry site of different viruses, and RNA elements of HIV. Full article

Flaviviruses (the genus Flavivirus of the Flaviviridae family) include many arthropod-borne viruses, often causing life-threatening diseases in humans, such as hemorrhaging and encephalitis. Although the flaviviruses have a significant clinical impact, it has become apparent that flavivirus replication is restricted by cellular factors induced by the interferon (IFN) response, which are called IFN-stimulated genes (ISGs). SHFL (shiftless antiviral inhibitor of ribosomal frameshifting) is a novel ISG that inhibits dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) infections. Interestingly, SHFL functions as a broad-spectrum antiviral factor exhibiting suppressive activity against various types of RNA and DNA viruses. In this review, we summarize the current understanding of the molecular mechanisms by which SHFL inhibits flavivirus infection and discuss the molecular basis of the inhibitory mechanism using a predicted tertiary structure of SHFL generated by the program AlphaFold2. Full article