Abstract
The shutoff of host protein synthesis by certain picornaviruses is mediated, at least in part, by proteolytic cleavage of eIF4G-1. Previously, we developed a cleavage site variant of eIF4G-1, termed eIF4G-1(SM), that was 100-fold more resistant to in vitro cleavage by Coxsackievirus 2A protease (2A(Pro)) than wild-type eIF4G-1 (eIF4G-1(WT)), but it was still digested at high protease concentrations. Here we identified a secondary cleavage site upstream of the primary site. We changed Gly at the P1'-position of the secondary site to Ala to produce eIF4G-1(DM). eIF4G-1(DM) was 1,000-10,000-fold more resistant to cleavage in vitro than eIF4G-1(WT). Full functional activity of eIF4G-1(DM) was demonstrated in vitro by its ability to restore cap-dependent translation to a 2A(Pro)-pretreated rabbit reticulocyte system. An isoform containing the binding site for poly(A)-binding protein, eIF4G-1e(DM), was more active in this assay than an isoform lacking it, eIF4G-1a(DM), but only with polyadenylated mRNA. Functional activity was also demonstrated in vivo with stably transfected HeLa cells expressing eIF4G-1(DM) from a tetracycline-regulated promoter. Cap-dependent green fluorescent protein synthesis was drastically inhibited by 2A(Pro) expression, but synthesis was almost fully restored by induction of either eIF4G-1a(DM) or eIF4G-1e(DM). By contrast, encephalomyocarditis virus internal ribosome entry site-dependent green fluorescent protein synthesis was stimulated by 2A(Pro); stimulation was suppressed by eIF4G-1e(DM) but not eIF4G-1a(DM).
Highlights
Upon infection of mammalian cells with picornaviruses of the rhino, aphtho, and enterovirus genera, most host protein synthesis is shut off coincident with the appearance of viral proteins [1]
Extracts from poliovirus-infected cells are unable to carry out cap-dependent translation, but this ability can be restored by addition of initiation factors from uninfected cells [2, 3], preparations containing eIF41 polypeptides [4, 5]. eIF4F, a complex of eIF4E, eIF4A, and eIF4G that is isolated from high salt extracts of ribosomes, was shown to restore cap-dependent translation in lysates of poliovirus-infected HeLa cells [6, 7]. eIF4F from uninfected cells stimulates cap-dependent but not cap-independent translation in the messenger RNA-dependent lysate (MDL) system, whereas the opposite is true for eIF4F from poliovirus-infected cells [8]
EIF4G was discovered as a result of its proteolysis coincident with the loss of cap-dependent initiation during poliovirus infection [9]. eIF4G is cleaved by the 2A protease (2APro) of Coxsackievirus B4 (CVB4) or human rhinovirus 2 (HRV2) into two functional domains, an N-terminal fragment that binds eIF4E and poly(A)-binding protein (PABP) and a C-terminal fragment that binds eIF4A and eIF3 (10 –12)
Summary
Site for PABP, but eIF4G-1a does not (Fig. 1). Cleavage of eIF4G-1 by 2APro was initially thought to be the major mechanism utilized by entero- and rhinoviruses to shut down host protein synthesis and allow viral protein synthesis to continue by IRES-driven translation [1]. Creating an eIF4G-1 variant that is resistant to 2APro would provide a useful tool to study the mechanism of host protein synthesis shutoff upon viral infection. Even though the variant (eIF4G-1aSM) was 100-fold more resistant to cleavage than eIF4G-1aWT, it was still digested, at sufficiently high protease concentrations and extended incubation times, into fragments of approximately the same size as those derived from eIF4G1aWT. This raised the question of whether cleavage occurred at the same or a different site. This variant is 1,000 –10,000-fold more resistant to CVB4 2APro than eIF4G-1WT in vitro. eIF4G-1DM was capable of restoring cap-dependent protein synthesis after 2APro expression both in vitro and in vivo
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