Search Results (15)

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

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

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

The interferon-induced host cell protein shiftless (SFL) was reported to inhibit human immunodeficiency virus (HIV) infection by blocking the –1 programmed ribosomal frameshifting (–1PRF) required for expression of the Gag-Pol polyprotein. However, it is not clear how SFL inhibits –1PRF. To address this question, we focused on a 36 amino acids comprising region (termed required for antiviral activity (RAA)) that is essential for suppression of –1PRF and HIV infection and is missing from SFL short (SFLS), a splice variant of SFL with unknown function. Here, we confirm that SFL, but not SFLS, inhibits HIV –1PRF and show that inhibition is cell-type-independent. Mutagenic and biochemical analyses demonstrated that the RAA region is required for SFL self-interactions and confirmed that it is necessary for ribosome association and binding to the HIV RNA. Analysis of SFL mutants with six consecutive amino-acids-comprising deletions in the RAA region suggests effects on binding to the HIV RNA, complete inhibition of –1PRF, inhibition of Gag-Pol expression, and antiviral activity. In contrast, these amino acids did not affect SFL expression and were partially dispensable for SFL self-interactions and binding to the ribosome. Collectively, our results support the notion that SFL binds to the ribosome and the HIV RNA in order to block –1PRF and HIV infection, and suggest that the multimerization of SFL may be functionally important. Full article

Since its initial characterization in 2016, the interferon stimulated gene Shiftless (SHFL) has proven to be a critical piece of the innate immune response to viral infection. SHFL expression stringently restricts the replication of multiple DNA, RNA, and retroviruses with an extraordinary diversity of mechanisms that differ from one virus to the next. These inhibitory strategies include the negative regulation of viral RNA stability, translation, and even the manipulation of RNA granule formation during viral infection. Even more surprisingly, SHFL is the first human protein found to directly inhibit the activity of the -1 programmed ribosomal frameshift, a translation recoding strategy utilized across nearly all domains of life and several human viruses. Recent literature has shown that SHFL expression also significantly impacts viral pathogenesis in mouse models, highlighting its in vivo efficacy. To help reconcile the many mechanisms by which SHFL restricts viral replication, we provide here a comprehensive review of this complex ISG, its influence over viral RNA fate, and the implications of its functions on the virus-host arms race for control of the cell. Full article

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use −1 programmed ribosomal frameshifting (−1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates −1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of −1 PRF in SARS-CoV-2 also inhibited −1 PRF in a range of bat CoVs—the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on −1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed −1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit −1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics. Full article

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (−1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro −1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential −1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting. Full article

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed “recoding”. In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for −1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed −1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed. Full article

A new mycovirus was found in the Fusarium culmorum strain A104-1 originally sampled on wheat in Belgium. This novel virus, for which the name Fusarium culmorum virus 1 (FcV1) is suggested, is phylogenetically related to members of the previously proposed family ‘’Unirnaviridae’’. FcV1 has a monopartite dsRNA genome of 2898 bp that harbors two large non-overlapping ORFs. A typical -1 slippery motif is found at the end of ORF1, advocating that ORF2 is translated by programmed ribosomal frameshifting. While ORF2 exhibits a conserved replicase domain, ORF1 encodes for an undetermined protein. Interestingly, a hypothetically transcribed gene similar to unirnaviruses ORF1 was found in the genome of Lipomyces starkeyi, presumably resulting from a viral endogenization in this yeast. Conidial isolation and chemical treatment were unsuccessful to obtain a virus-free isogenic line of the fungal host, highlighting a high retention rate for FcV1 but hindering its biological characterization. In parallel, attempt to horizontally transfer FcV1 to another strain of F. culmorum by dual culture failed. Eventually, a screening of other strains of the same fungal species suggests the presence of FcV1 in two other strains from Europe. Full article

In contrast to −1 programmed ribosomal frameshifting (PRF) stimulation by an RNA pseudoknot downstream of frameshifting sites, a refolding upstream RNA hairpin juxtaposing the frameshifting sites attenuates −1 PRF in human cells and stimulates +1 frameshifting in yeast. This eukaryotic functional mimicry of the internal Shine-Dalgarno (SD) sequence-mediated duplex was confirmed directly in the 70S translation system, indicating that both frameshifting regulation activities of upstream hairpin are conserved between 70S and 80S ribosomes. Unexpectedly, a downstream pseudoknot also possessed two opposing hungry codon-mediated frameshifting regulation activities: attenuation of +1 frameshifting and stimulation of a non-canonical −1 frameshifting within the +1 frameshift-prone CUUUGA frameshifting site in the absence of release factor 2 (RF2) in vitro. However, the −1 frameshifting activity of the downstream pseudoknot is not coupled with its +1 frameshifting attenuation ability. Similarly, the +1 frameshifting activity of the upstream hairpin is not required for its −1 frameshifting attenuation function Thus, each of the mRNA duplexes flanking the two ends of a ribosomal mRNA-binding channel possesses two functions in bi-directional ribosomal frameshifting regulation: frameshifting stimulation and counteracting the frameshifting activity of each other. Full article

5-fluorouracil (5-FU) is a chemotherapeutic agent that has been extensively studied since its initial development in the 1950s. It has been suggested that the mechanism of action of 5-FU involves both DNA- and RNA-directed processes, but this has remained controversial. In this study, using a series of in vivo reporter constructs capable of measuring translational recoding, we demonstrate that cells exposed to 5-FU display a reduced capacity to engage in a variety of translational recoding events, including +1 programmed frameshifting (PRF) and −1 PRF. In addition, 5-FU-treated cells are much less accurate at stop codon recognition, resulting in a significant increase in stop codon-readthrough. Remarkably, while the efficiency of cap-dependent translation appears to be unaffected by 5-FU, 5-FU-treated cells display a decreased ability to initiate cap-independent translation. We further show that knockdown of thymidylate synthase, an enzyme believed to be at the center of 5-FU-induced DNA damage, has no effect on the observed alterations in translational recoding. On the other hand, ribosomal RNA (rRNA) pseudouridylation, which plays an important role in translational recoding, is significantly inhibited. Taken together, our results suggest that the observed effect of 5-FU on recoding is an RNA-directed effect. Our results are the first to show definitely and quantitatively that translational recoding is affected by exposure to 5-FU. Thus, it is possible that a substantial portion of 5-FU cytotoxicity might possibly be the result of alterations in translational recoding efficiency. Full article

Two novel double-stranded RNA (dsRNA) mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2) and Fusarium poae dsRNA virus 3 (FpV3), were isolated from the plant pathogenic fungus, Fusarium poae strain SX63, and molecularly characterized. FpV2 and FpV3, with respective genome sequences of 9518 and 9419 base pairs (bps), are both predicted to contain two discontinuous open reading frames (ORFs), ORF1 and ORF2. A hypothetical polypeptide (P1) and a RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively. Phytoreo_S7 domain (pfam07236) homologs were detected downstream of the RdRp domain (RdRp_4; pfam02123) of the ORF2-coded proteins of both FpV2 and FpV3. The same shifty heptamers (GGAAAAC) were both found immediately before the stop codon UAG of ORF1 in FpV2 and FpV3, which could mediate programmed –1 ribosomal frameshifting (–1 PRF). Phylogenetic analysis based on RdRp sequences clearly place FpV2 and FpV3 in a taxonomically unassigned dsRNA mycovirus group. Together, with a comparison of genome organization, a new taxonomic family termed Fusagraviridae is proposed to be created to include FpV2- and FpV3-related dsRNA mycoviruses, within which FpV2 and FpV3 would represent two distinct virus species. Full article