In a previous study, it was demonstrated that although MV infection induces significant host shutoff, infected cells contain normal levels of host mRNA (unpublished data). This suggests that MV-induced shutoff occurs at the level of host translation. In most eukaryotic mRNAs, translation initiation commences with the recruitment of cap binding protein complex, eIF4F, composed of eIF4E (cap binding protein), eIF4A, and eIF4G, to the capped 5′ end (6
). The 40S ribosomal subunit, which carries eIF3 and the ternary initiator tRNAiMet
-eIF2-GTP complex, is then recruited to the 5′ end of the mRNA through interactions between eIF3 and eIF4G (6
). The 40S subunits scan the mRNA in a 5′-to-3′ direction until an appropriate start codon is encountered. At this point, the anticodon in initiator tRNA (
), positioned in the ribosomal P site, engages in base pairing with the start codon in the mRNA. The large ribosomal 60A subunit joins and protein synthesis commences (6
). It is known that several RNA viruses disrupt host translation initiation as part of their selective translation strategies. These viruses primarily target the eIF4F complex and related auxiliary factors in order to inhibit host translation. For example, during picornavirus infection, eIF4G is cleaved by virus-encoded proteases (5
). In cells infected with EMCV and poliovirus, an eIF4E-binding protein (4E-BP) is dephosphorylated, resulting in sequestration of eIF4E by 4E-BP (3
). During coxsackievirus and poliovirus infection, the poly(A)-binding protein, which forms a closed-loop translation complex in conjunction with eIF4G, is proteolyzed (8
). In influenza virus-infected cells, eIF4E is partially inactivated by dephosphorylation (2
). Our preliminary studies indicate that no modifications of the eIF4F complex or any related auxiliary factors are observed during MV infection (unpublished data). In the present study, however, we show that MV-N has the ability to bind to eIF3-p40 and suppress mRNA translation in vitro (Fig. and ). Binding was necessary for suppression since the MV-N deletion clone, which does not interact with eIF3-p40, scarcely inhibited translation reactions (Fig. ). These results indicate that MV-N uniquely suppresses translation reactions by binding to eIF3-p40. Our preliminary studies revealed that 293 and Cos-7 cells, which overexpressed SLAM (a receptor for wild-type MV) by plasmid transfection, permitted wild-type MV infection and replication (unpublished data). This implies that the results observed in Cos-7 cells (Fig. and ) and 293-MVN cells (Fig. ) reflect the phenomenon during MV infection. To further confirm the effect of MV-N on translation in MV-infected cells, we attempted to generate a recombinant MV that possesses mutant N protein lacking the binding site to eIF3-p40 using reverse genetics. However, the variant virus could not be rescued by several trials using conditions with which other recombinant MVs were well rescued (data not shown). This result implied that the amino acid residues that participate in interaction with eIF3-p40 may also be important for the other function of N protein.
Mammalian eIF3 is the largest (650 kDa) of all initiation factors and is composed of 11 or 12 different subunits whose precise patterns of interaction with other factors and stoichiometry remain poorly understood. To analyze the binding manner of MV-N on eIF3 complex, we attempted to immunoprecipitation assay using antibodies against several subunits of eIF3, but they did not pull down the whole eIF3 complex effectively, and the exact interaction between MV-N and eIF3 complex could not be defined (data not shown). Recent reports have revealed that a viral protein and several host factors regulate host protein synthesis by modulating eIF3 function. For example, the virus- and interferon (IFN)-inducible human protein, p56, binds to the p48 subunit of eIF3 and inhibits in vitro translation and cellular protein synthesis (4
). Mouse p56 also inhibits in vitro translation by binding to the p110 subunit of eIF3 (4
). Cyclin-dependent kinase 11 interacts with the p47 subunit of eIF3 and suppresses translation by phosphorylating a specific serine residue in the p47 subunit (24
). In Norwalk virus, the RNA genome-linked protein VPg binds to the p66 subunit of eIF3 and inhibits in vitro translation reactions (1
), which are thought to initiate protein synthesis from viral RNA. Our present study reveals a new mode of mammalian protein synthesis regulation that involves the modulation of eIF3 function via the p40 subunit.
GST-N inhibited both cap-dependent and EMCV-IRES-mediated translation in a dose-dependent manner (Fig. ). The EMCV-IRES requires canonical eIFs, including eIF3, in order to initiate translation (18
). Stable binding of eIF3 to the IRES is essential for the attachment of the 40S subunit to the IRES (26
). This suggests that the suppression of EMCV-IRES-mediated translation by MV-N may occur through binding of MV-N to eIF3-p40, as seen in cap-dependent translation. In contrast, IGR-IRES-mediated translation is apparently insensitive to the inhibitory effects of GST-N (Fig. ). The IGR-IRES can assemble 80S ribosomes from purified 40S and 60S ribosomal subunits in the absence of eIF2,
, or GTP hydrolysis and without any known canonical eIFs (29
). Thus, eIF3 is not involved in IGR-IRES/40S subunit assembly. These unique characteristics may explain why IGR-IRES-mediated translation is only modestly inhibited by GST-N. At high concentrations of GST-N, slight suppression of IGR-IRES-mediated translation is observed; however, this may result because endogenous initiation factors are still be present in the lysates, and binding of MV-N to endogenous eIF3 may be interfering with 40S subunit binding to IGR-IRES.
MV-N induced the suppression of protein synthesis at the level of translation both in vitro and in whole cells (Fig. ). However, suppression in whole cells was partial, and suppression rates reached a plateau at ca. 50 to 60% inhibition. Our preliminary data indicate that MV-infected cells suppress more than 90% of host protein synthesis at 36 h postinfection (unpublished data), suggesting that one or more additional pathways are involved in MV-mediated host shutoff. Previous reports on other viral infections show that type I IFN induced by infection activates multiple pathways to shutoff host function, including phosphorylation of eIF2α by double-stranded RNA-activated protein kinase (21
), activation of RNase L and subsequent degradation of mRNA by 2′,5′-oligoadenylate synthetase (27
), and induction of virus-inducible protein P56 which binds to eIF3-p48 (4
). However, a previous report showed that the suppression of overall cellular protein synthesis by IFN is not complete but rather only ca. 50% inhibition (4
). Indeed, we have shown that MV infection induces phosphorylation of eIF2α, but its participation for the inhibition of host protein synthesis in the overall host shutoff is partial and restricted at an early phase of infection (Inoue et al., unpublished). These data indicate that significant host shutoff induced by MV may occur through multiple cooperating pathways.
In addition to the significant suppression of host translation, we have confirmed that the selective translation of viral mRNA still occurs in MV-infected cells (unpublished data). Recently, our preliminary study identified a host factor that is implicated in the preferential translation of MV mRNAs in infected cells (unpublished data). From these data, it can be speculated that MV utilizes advanced strategies that control the host cells while evade MV itself from shutoff state in the infected cells.