Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

Abstract

Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5= untranslated region (5=UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCEThe nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection. Hand-foot-and-mouth disease (HFMD) is a common viral illness in infants and children. The disease causes fever and skin rash or blister-like eruptions (1). HFMD is caused by viruses that belong to the Enterovirus genus of the Picornavirus family, which mainly includes coxsackieviruses and echoviruses (1, 2). Enteroviruses, especially enterovirus 71 (EV71), have been associated with HFMD and may lead to disease with severe effects on morbidity and mortality (3) and a high rate of neurological complications, such as aseptic meningitis, acute flaccid paralysis, and fatal neurogenic pulmonary edema (4, 5). The first EV71 strain (BrCr-CA-70) was isolated in 1970 in California and reported ...

Sam68 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 2 of Viral 5′ Untranslated Region

“…Since the function of SL2 of the HCV 5= UTR in regulating HCV life cycle focuses on either regulation or translation, based on its different binding region, we ought to explore the narrow region of SL2 that connects to Sam68 in a future study. Previous reports show that the KH domain of Sam68 is required for the interaction of RNA virus (33). Our data support that the RGG box domain is necessary for the interaction between Sam68 and the HCV genome.…”

“…Our data showed that Sam68 is not involved in HCV RNA translation. Upon HCV infection, Sam68 relocated from the nucleus to the cytoplasm to implement its interaction of the HCV genome, which conjuncts with the discovery that enterovirus 71 (EV71) and FMDV induce the redistribution of Sam68 (23,33). Interestingly, we observed a weak interaction between Sam68 and PCBP2 in physiology, and the association appeared as upregulation upon HCV infection.…”

Porcine epidemic diarrhea virus ORF3 protein causes endoplasmic reticulum stress to facilitate autophagy

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Enterovirus 71 inhibits cellular type I interferon signaling by inhibiting host RIG-I ubiquitination

“…EV71 (BrCr-Tr strain; GenBank accession number AB204852.1) infection was carried out as described previously [20,21]. In brief, RD cells were cultured in FBS-free medium and infected with EV71 at the multiplicity of infection (MOI) indicated in the figure legends.…”

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