Insights from structural studies of the Cardiovirus 2A protein

Cardioviruses are single-stranded RNA viruses of the family Picornaviridae. In addition to being the first example of internal ribosome entry site 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.

Expanding uncapped translation and emerging function of circular RNA in carcinomas and noncarcinomas

Circular RNAs (circRNAs) are classified as noncoding RNAs because they are devoid of a 5’ end cap and a 3’ end poly (A) tail necessary for cap-dependent translation. However, increasing numbers of translated circRNAs identified through high-throughput RNA sequencing overlapping with polysome profiling indicate that this rule is being broken. CircRNAs can be translated in cap-independent mechanism, including IRES (internal ribosome entry site)-initiated pattern, MIRES (m6A internal ribosome entry site) -initiated patterns, and rolling translation mechanism (RCA). CircRNA-encoded proteins harbour diverse functions similar to or different from host proteins. In addition, they are linked to the modulation of human disease including carcinomas and noncarcinomas. CircRNA-related translatomics and proteomics have attracted increasing attention. This review discusses the progress and exclusive characteristics of circRNA translation and highlights the latest mechanisms and regulation of circRNA translatomics. Furthermore, we summarize the extensive functions and mechanisms of circRNA-derived proteins in human diseases, which contribute to a better understanding of intricate noncanonical circRNA translatomics and proteomics and their therapeutic potential in human diseases.

Insights into the secondary and tertiary structure of the Bovine Viral Diarrhea Virus Internal Ribosome Entry Site

The Internal Ribosome Entry Site (IRES) RNA of Bovine viral diarrhea virus (BVDV), an economically significant Pestivirus, is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modeling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA forms in solution a modular architecture composed of three distinct structural domains (I-III). Two regions within domain III are engaged in tertiary interactions to form an H-type pseudoknot. Computational modeling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.

Resurrection of a Viral Internal Ribosome Entry Site from a 700 Year Old Ancient Northwest Territories Cripavirus

The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented, streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. The aNCV IGR sequence adopts a secondary structure similar to contemporary IGR IRES structures, however, there are subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IRES could bind to purified ribosomes, and toeprinting analysis pinpointed the start site at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR can direct translation in vitro in a PKI-dependent manner. Lastly, a chimeric infectious clone swapping in the aNCV IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework for the importance of this viral translational mechanism.

Resurrection of a viral internal ribosome entry site from a 700 year old ancient Northwest Territories cripavirus

ABSTRACTThe dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. Structural prediction of the aNCV IGR sequence generated a secondary structure similar to contemporary IGR IRES structures. There are, however, subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IGR IRES could bind to purified salt-washed human ribosomes and compete with a prototypical IGR IRES for ribosomes. Toeprinting analysis using primer extension pinpointed the putative start site of the aNCV IGR at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR IRES can direct internal ribosome entry in vitro in a manner dependent on the integrity of the PKI domain. Lastly, we generated a chimeric virus clone by swapping the aNCV IRES into the cricket paralysis virus infectious clone. The chimeric infectious clone with an aNCV IGR IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework in the importance of this viral translational mechanism.IMPORTANCEInternal ribosome entry sites are RNA structures that are used by some positive-sense monopartite RNA viruses to drive viral protein synthesis. The origin of internal ribosome entry sites is not known. Using biochemical approaches, we demonstrate that an RNA structure from an ancient viral genome that was discovered from a 700-year-old caribou feces trapped in subarctic ice is functionally similar to modern internal ribosome entry sites. We resurrect this ancient RNA mechanism by demonstrating that it can support virus infection in a contemporary virus clone, thus providing insights into the origin and evolution of this viral strategy.

The internal ribosome entry site of the Dengue virus mRNA is active when cap-dependent translation initiation is inhibited.

Dengue virus (DENV) is an enveloped, positive-sense, single-stranded RNA virus belonging to the Flaviviridae family. Translation initiation of the DENV mRNA can occur following a cap-dependent or a cap-independent mechanism. Two non-mutually exclusive cap-independent mechanisms of translation initiation have been described for the DENV mRNA. The first corresponds to a 5′end-dependent internal ribosome entry site (IRES)-independent mechanism, while the second relies on IRES-dependent initiation. In this report, we study the recently discovered DENV IRES. Results show that the DENV IRES is functional in the rabbit reticulocyte (RRL) in vitro translation system. In accordance, the activity of DENV IRES was resistant to the cleavage of eIF4G by the Foot-and-mouth disease virus leader protease in RRL. In cells, the DENV IRES exhibited only a marginal activity under standard culture conditions. The DENV IRES showed weak activity in HEK 293T cells; however, the DENV IRES activity was significantly enhanced in HEK 293T cells expressing the Human rhinovirus 2A protease. These findings suggest that the DENV IRES enables viral protein synthesis under conditions that suppress canonical translation initiation. IMPORTANCE Dengue virus (DENV), the etiological agent of Dengue, a febrile and hemorrhagic disease, infects millions of people per year in tropical and subtropical countries. When infecting cells, DENV induces stress conditions known to inhibit canonical protein synthesis. Under these conditions, DENV mRNA thrives using non-canonical modes of translation initiation. In this study, we characterize the mechanism dependent upon an internal ribosome entry site (IRES). Herein, we describe the activity of the DENV IRES in vitro and cells. We show that in cells, DENV IRES enables the viral mRNA to translate under conditions that suppress canonical translation initiation.

Custom-designed, degradation-resistant messenger RNAs in yeast

ABSTRACTStructured RNA elements that protect RNA transcripts from 5’→3’ degradation are becoming useful research tools. Here we show that exonuclease-resistant RNA structures (xrRNAs) from Flaviviruses can be used to protect heterologous messenger RNAs (mRNAs) from 5’→3’ degradation in budding yeast. Installation of xrRNAs ahead of a downstream internal ribosome entry site (IRES) leads to the accumulation of partially-degraded mRNAs that are substrates for cap-independent translation of a LacZ reporter, yielding a 30-fold increase in measured β-galactosidase activity. Additionally, by monitoring the translation of dual-luciferase reporters we show that xrRNA sequences do not interfere with the progression of an elongating ribosome. Combined these data indicate that xrRNA elements can be used in creative ways to stabilize RNAs with potentially useful applications.