PMC:4576194 / 18934-23102 JSONTXT

{"project":"2_test","denotations":[{"id":"26266418-22795973-144197541","span":{"begin":119,"end":120},"obj":"22795973"},{"id":"26266418-24499791-144197542","span":{"begin":471,"end":473},"obj":"24499791"},{"id":"26266418-25827528-144197543","span":{"begin":594,"end":596},"obj":"25827528"},{"id":"26266418-24499791-144197544","span":{"begin":879,"end":881},"obj":"24499791"},{"id":"26266418-18448518-144197545","span":{"begin":1843,"end":1845},"obj":"18448518"},{"id":"26266418-24974120-144197546","span":{"begin":1982,"end":1984},"obj":"24974120"},{"id":"26266418-22476100-144197547","span":{"begin":2102,"end":2104},"obj":"22476100"},{"id":"26266418-22628567-144197548","span":{"begin":2105,"end":2107},"obj":"22628567"},{"id":"26266418-21079685-144197549","span":{"begin":2116,"end":2118},"obj":"21079685"},{"id":"26266418-23637406-144197550","span":{"begin":2129,"end":2131},"obj":"23637406"},{"id":"26266418-18501944-144197551","span":{"begin":2350,"end":2352},"obj":"18501944"},{"id":"26266418-16386773-144197552","span":{"begin":2353,"end":2355},"obj":"16386773"},{"id":"26266418-19131084-144197553","span":{"begin":2356,"end":2358},"obj":"19131084"},{"id":"26266418-11714969-144197554","span":{"begin":2438,"end":2440},"obj":"11714969"},{"id":"26266418-16927014-144197555","span":{"begin":2441,"end":2443},"obj":"16927014"},{"id":"26266418-11714969-144197556","span":{"begin":2522,"end":2524},"obj":"11714969"},{"id":"26266418-16927014-144197557","span":{"begin":2727,"end":2729},"obj":"16927014"},{"id":"26266418-16554823-144197558","span":{"begin":3150,"end":3152},"obj":"16554823"},{"id":"26266418-23637406-144197559","span":{"begin":3416,"end":3418},"obj":"23637406"},{"id":"26266418-9261361-144197560","span":{"begin":3419,"end":3421},"obj":"9261361"},{"id":"26266418-17626087-144197561","span":{"begin":3422,"end":3424},"obj":"17626087"}],"text":"3. Discussion\nThe CSFV NS5A functions as the modulation of viral RNA replication via interacting with NS5B and 3ʹ-UTR [8]. In addition, the NS5A protein can inhibit the IRES-mediated translation through binding to the IRES located in the 5ʹ-UTR. A previous study showed that the NS5A-interacting cellular proteins were screened using Y2H analysis, and the identified proteins are mostly related to gene transcription, protein degradation, protein folding and metabolism [26]. Recently, heat shock protein 70 has been identified to interact with the CSFV NS5A and enhance viral RNA replication [27]. In this study, eEF1A was identified as a novel NS5A partner and a negative regulator of CSFV replication. eEF1A had not been identified in the previous study, probably due to the different cell types (endothelial cells vs. primary macrophages) used to construct the cDNA library [26]. We showed that NS5A and eEF1A were colocalized in the cytoplasm. As NS5A is the component of the replication complex, whether eEF1A is also localized in the replication complex needs further investigation.\nThe domain I of eEF1A was shown to be critical for the NS5A–eEF1A interaction, implying that it may be a potential target of anti-CSFV strategies. We also showed that eEF1A was able to bind to the IRES located in the 5ʹ-UTR and inhibit its activity. In addition, we investigated the effects of eEF1A on the inhibition of the IRES activity by NS5A; however, we demonstrated that eEF1A did not antagonize the inhibition of the IRES activity by NS5A, indicating that NS5A and eEF1A perhaps bind to distinct parts of the IRES.\nSeveral viral proteins have been observed to associate with eEF1A. The nucleocapsid protein of severe acute respiratory syndrome coronavirus (SARS-CoV) could modulate cell cytokinesis and proliferation via interacting with eEF1A [28]. eEF1A also interacts with the nucleocapsid protein of transmissible gastroenteritis virus (TGEV) and regulates the virus replication [29]. Furthermore, eEF1A can be utilized as the cofactors of the viral RNA synthesis for several viruses, such as HIV-1 [22,23], TBSV [21] and WNV [30]. Previous studies on turnip mosaic virus (TuMV), tobacco mosaic virus (TMV) and TBSV have shown that eEF1A interacts primarily with the viral RdRp complex, and thus acts as a chaperone and supports viral replication [31,32,33]. As members of Flaviviridae, both BVDV NS5A and HCV NS4A interact with eEF1A [24,34]. Thereinto, the exact role of eEF1A on the BVDV replication remains unclear [24]. The HCV NS4A inhibits the viral IRES-mediated translation, and the translation inhibitory effect of NS4A was relieved by the addition of purified recombinant eEF1A in the in vitro translation system [34]. However, we showed here that eEF1A negatively regulates the CSFV growth. We conclude that the different roles of eEF1A in viral replication may vary depending on the distinct interactions between eEF1A and different viruses. In this study, the eEF1A was shown to bind to the IRES and inhibit its activity, thus it is possible to suppress the CSFV replication.\nSince eEF1A binds to aa-tRNA among mammalian RNA species [35], it is possible for eEF1A to bind to the similar RNA structures located in 5ʹ- or 3ʹ-UTR of the viral genome. It has been shown that interaction between eEF1A and the 3ʹ-terminal stem-loop of the WNV genomic RNA facilitates the viral minus-strand RNA synthesis [30,36,37]. In the present study, we demonstrated that eEF1A binds to IRES located in the 5ʹ-UTR of CSFV. Whether eEF1A binds to 3ʹ-UTR of CSFV and influences RNA replication requires further study. We showed that eEF1A inhibits CSFV growth and reduces IRES activity of CSFV. Thus, we speculate that eEF1A affects the process of the viral protein translation via interacting with the CSFV IRES.\nIn summary, we demonstrated that eEF1A interacts with NS5A and negatively modulates the CSFV growth. The possible mechanism of the negative effect of eEF1A on the CSFV growth is that eEF1A interacts with the CSFV IRES and inhibits its activity. Further studies are needed to define the detailed interplay among eEF1A, NS5A and IRES in the life cycle of CSFV."}