Translation of Viral mRNA without Active eIF2: The Case of Picornaviruses

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain.
PLoS ONE (Impact Factor: 3.23). 07/2011; 6(7):e22230. DOI: 10.1371/journal.pone.0022230
Source: PubMed


Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.

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Available from: Miguel Angel Sanz, Oct 04, 2015
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    • "In this sense, a dual mechanism is responsible for picornavirus mRNA translation. At early times of infection picornavirus mRNA is translated following a canonical mechanism that employs intact eIF4G and active eIF2, whereas at late times inactivation of eIF2 does not abrogate viral protein synthesis [14]. Moreover, synthesis of PV 2Apro at high levels in culture cells makes translation of mRNAs containing EMCV or PV IRESs independent of eIF2 [15]. "
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    ABSTRACT: Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A(pro) or L(pro). The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A(pro) strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L(pro) strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A(pro) and L(pro) exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L(pro). Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.
    PLoS ONE 12/2012; 7(12):e52065. DOI:10.1371/journal.pone.0052065 · 3.23 Impact Factor
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    • "Another translation initiation factor eIF4GII as well as the Poly-(A) binding protein (PABP), a protein facilitating the formation of a closed translation initiation loop by interaction of the 5 and 3 ends of the mRNA, has been reported to be cleaved by picornaviral 2A (Gradi et al., 1998; Joachims et al., 1999). All these cleavages often correspond with a translational shift to IRES-dependent translation (Redondo et al., 2011; Welnowska et al., 2011). Another group also showed that the shift in translation seen during the later phase of poliovirus infection is not entirely due to phosphorylation (inactivation) of eIF2α (see Discussion in later session), but may also depend upon protease 3C activation and cleavage of another translation initiation factor, eIF5B, to a C-terminal truncated version thought to replace eIF2 during translation (White et al., 2011). "
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    ABSTRACT: Many virus infections and stresses can induce endoplasmic reticulum (ER) stress response, a host self-defense mechanism against viral invasion and stress. During this event, viral and cellular gene expression is actively regulated and often encounters a switching of the translation initiation from cap-dependent to internal ribosome-entry sites (IRES)-dependent. This switching is largely dependent on the mRNA structure of the 5' untranslated region (5' UTR) and on the particular stress stimuli. Picornaviruses and some other viruses contain IRESs within their 5' UTR of viral genome and employ an IRES-driven mechanism for translation initiation. Recently, a growing number of cellular genes involved in growth control, cell cycle progression and apoptosis were also found to contain one or more IRES within their long highly structured 5' UTRs. These genes initiate translation usually by a cap-dependent mechanism under normal physiological conditions; however, in certain environments, such as infection, starvation, and heat shock they shift translation initiation to an IRES-dependent modality. Although the molecular mechanism is not entirely understood, a number of studies have revealed that several cellular biochemical processes are responsible for the switching of translation initiation to IRES-dependent. These include the cleavage of translation initiation factors by viral and/or host proteases, phosphorylation (inactivation) of host factors for translation initiation, overproduction of homologous proteins of cap-binding protein eukaryotic initiation factors (eIF)4E, suppression of cap-binding protein eIF4E expression by specific microRNA, activation of enzymes for mRNA decapping, as well as others. Here, we summarize the recent advances in our understanding of the molecular mechanisms for the switching of translation initiation, particularly for the proteins involved in cell survival and apoptosis in the ER stress pathways during viral infections.
    Frontiers in Microbiology 03/2012; 3:92. DOI:10.3389/fmicb.2012.00092 · 3.99 Impact Factor
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    • "A PV replicon (pRLuc31) containing the luciferase (luc) gene replacing the viral structural proteins was used [38]. As controls, cells were also electroporated with Cap-luc or CrPV IGR-luc mRNAs [28], [29] and at 1 hpe cells were treated with different concentrations of Ars (0, 50, 100 and 200 µM) for 1 h. Electroporation of these RNAs into BHK-21 cells gives rise to luc synthesis from the beginning of transfection. "
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    ABSTRACT: Poliovirus RNA utilizes eIF2 for the initiation of translation in cell free systems. Remarkably, we now describe that poliovirus translation takes place at late times of infection when eIF2 is inactivated by phosphorylation. By contrast, translation directed by poliovirus RNA is blocked when eIF2 is inactivated at earlier times. Thus, poliovirus RNA translation exhibits a dual mechanism for the initiation of protein synthesis as regards to the requirement for eIF2. Analysis of individual poliovirus non-structural proteins indicates that the presence of 2A(pro) alone is sufficient to provide eIF2 independence for IRES-driven translation. This effect is not observed with a 2A(pro) variant unable to cleave eIF4G. The level of 2A(pro) synthesized in culture cells is crucial for obtaining eIF2 independence. Expression of the N-or C-terminus fragments of eIF4G did not stimulate IRES-driven translation, nor provide eIF2 independence, consistent with the idea that the presence of 2A(pro) at high concentrations is necessary. The finding that 2A(pro) provides eIF2-independent translation opens a new and unsuspected area of research in the field of picornavirus protein synthesis.
    PLoS ONE 10/2011; 6(10):e25699. DOI:10.1371/journal.pone.0025699 · 3.23 Impact Factor
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