No abstract

A diverse group of viruses subvert the host translational machinery to promote viral genome translation. This process often involves altering canonical translation initiation factors to repress cellular protein synthesis while viral proteins are efficiently synthesized. The discovery of this strategy in picornaviruses, which is based on the use of internal ribosome entry site (IRES) elements, opened new avenues to study alternative translational control mechanisms evolved in different groups of RNA viruses. IRESs are cis-acting RNA sequences that adopt three-dimensional structures and recruit the translation machinery assisted by a subset of translation initiation factors and various RNA binding proteins. However, IRESs present in the genome of different RNA viruses perform the same function despite lacking conservation of primary sequence and secondary RNA structure, and differing in host factor requirement to recruit the translation machinery. Evolutionary conserved motifs tend to preserve sequences impacting on RNA structure and RNA-protein interactions important for IRES function. While some motifs are found in various picornavirus IRESs, others occur only in one type reflecting specialized factor requirements. This review is focused to describe recent advances on the principles and RNA structure features of picornavirus IRESs.

In eukaryotic cells translation initiation occurs through two alternative mechanisms, a cap-dependent operating in the majority of mRNAs, and a 5′-end-independent driven by internal ribosome entry site (IRES) elements, specific for a subset of mRNAs. IRES elements recruit the translation machinery to an internal position in the mRNA through a mechanism involving the IRES structure and several trans-acting factors. Here, we identified Gemin5 protein bound to the foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) IRES using two independent approaches, riboproteomic analysis and immunoprecipitation of photocroslinked factors. Functional analysis performed in Gemin5 shRNA-depleted cells, or in in vitro translation reactions, revealed an unanticipated role of Gemin5 in translation control as a down-regulator of cap-dependent and IRES-driven translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also functions as a modulator of translation activity.

Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-␣), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV. IMPORTANCEViral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness. Selection of viral mutants resistant to antiviral agents is a major problem for the successful treatment of viral diseases. In the case of RNA viruses, high mutation rates during genome replication provide viral populations with an ample reservoir of phenotypic variants, including mutants that can escape selective constraints. Resistance to a single drug that targets a viral protein develops at a rate that depends on the genetic barrier (number and types of mutations needed to acquire resistance) and the phenotypic barrier (fitness cost) imposed by the resistance mutations (1-16). When drug resistance mutations do not entail a significant fitness cost-either because the mutations per se do not critically affect viral functions or because compensatory mutations are acquired-they may reach detectable levels despite no prior exposure of the viral population to the drug (1, 16-27).Control of hepatitis C virus (HCV) infections is hampered by the complexity of HCV quasispecies replicating in the liver (16,28,29). Directly acting antiviral agents (DAAs)-some currently in use and others under development-offer great promise for control of HCV either as a substitute for or complement of the standard-of-care (SOC) therapy based on treatment using a combination of pegylated alpha interferon (IFN-␣) and ribavirin (30)(31)(32)(33)(34)(35)(36). Combinations that include the polymerase inhibitor sofosbuvir have produced sustained viral responses that in some cases have been higher than 90% in clinical trials (37-40), but the possible impact of resistance mutations is not known; sofosbuvir resistance substitution S282T in NS5B is present in the ...

No abstract

Little is known about the tertiary structure of internal ribosome entry site (IRES) elements. The central domain of foot-andmouth disease (FMDV) IRES, named 3 or I, contains a conserved GNRA motif, essential for IRES activity. We have combined functional analysis with RNA probing to define its structural organization. We have found that a UNCG motif does not functionally substitute the GNRA motif; moreover, binding of synthetic GNRA stem-loops to domain 3 was significantly reduced in RNAs bearing UCCG or GUAG substitutions. The apical region of domain 3 consists of a four-way junction where residues of the GNRA tetraloop are responsible for the organization of the adjacent stem-loops, as deduced from ribonucleases and dimethyl sulfate accessibility. A single A-to-G substitution in the fourth position of this motif led to a strong RNA reorganization, affecting several nucleotides away in the secondary structure of domain 3. The study of mutants bearing UNCG or GUAG tetraloops revealed lack of protection to chemical attack in native RNA at specific nucleotides relative to the parental GUAA, suggesting that the GNRA motif dictates the organization and stability of domain 3. This effect is likely mediated by the interaction with distant residues. Therefore, the GNRA motif plays a crucial role in the organization of IRES structure with important consequences on activity.

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 39 region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.

The untranslated regions (UTRs) of the foot-and-mouth disease virus (FMDV) genome contain multiple functional elements. In the 59 UTR, the internal ribosome entry site (IRES) element governs cap-independent translation initiation, whereas the S region is presumably involved in RNA replication. The 39 UTR, composed of two stem-loops and a poly(A) tract, is required for viral infectivity and stimulates IRES activity. Here, it was found that the 39 end established two distinct strand-specific, long-range RNA-RNA interactions, one with the S region and another with the IRES element. These interactions were not observed with the 39 UTR of a different picornavirus. Several results indicated that different 39 UTR motifs participated in IRES or S region interactions. Firstly, a high-order structure adopted by both the entire IRES and the 39 UTR was essential for RNA interaction. In contrast, the S region interacted with each of the stem-loops. Secondly, S-39 UTR interaction but not IRES-39 UTR interaction was dependent on a poly(A)-dependent conformation. However, no other complexes were observed in mixtures containing the three transcripts, suggesting that these regions did not interact simultaneously with the 39 UTR probe. Cellular proteins have been found to bind the S region and one of these also binds to the 39 UTR in a competitive manner. Our data suggest that 59-39-end bridging through both direct RNA-RNA contacts and RNA-protein interactions may play an essential role in the FMDV replication cycle.