Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.

Picornavirus Type 1 IRESs comprise five principal domains (dII-dVI). Whereas dV binds eIF4G, a conserved AUG in dVI was suggested to stimulate attachment of 43S ribosomal preinitiation complexes, which then scan to the initiation codon. Initiation on Type 1 IRESs also requires IRES trans-acting factors (ITAFs), and several candidates have been proposed. Here, we report the in vitro reconstitution of initiation on three Type 1 IRESs: poliovirus (PV), enterovirus 71 (EV71), and bovine enterovirus (BEV). All of them require eIF2, eIF3, eIF4A, eIF4G, eIF4B, eIF1A, and a single ITAF, poly(C) binding protein 2 (PCBP2). In each instance, initiation starts with binding of eIF4G/eIF4A. Subsequent recruitment of 43S complexes strictly requires direct interaction of their eIF3 constituent with eIF4G. The following events can differ between IRESs, depending on the stability of dVI. If it is unstructured (BEV), all ribosomes scan through dVI to the initiation codon, requiring eIF1 to bypass its AUG. If it is structured (PV, EV71), most initiation events occur without inspection of dVI, implying that its AUG does not determine ribosomal attachment.

During the acute phase response (APR), cytokines induce marked alterations in lipid metabolism including an increase in serum triglycerides, a decrease in hepatic fatty acid oxidation, a decrease in bile acid synthesis, and a decrease in HDL. Here we demonstrate that TNF and IL-1, but not IL-6, decrease the expression of RXRα, PPARα, PPARγ, LXRα and coactivators PGC-1α, PGC-1β and SRC-1 in Hep3B human hepatoma cells. Additionally, treatment of mice with TNF and IL-1 also decreased RXRα, PPARα, PPARγ, LXRα, and PGC-1α mRNA levels in the liver. These decreases were accompanied by reduced binding of nuclear extracts to RXR, PPAR, and LXR response elements and decreased luciferase activity driven by PPAR and LXR response elements. In addition, the mRNA levels of proteins regulated by PPARα (CPT 1α) and LXR (SREBP-1c) were decreased in Hep 3B cells treated with TNF or IL-1. Finally, using constructs of the LXRα promoter or the PGC-1α promoter linked to luciferase we were able to demonstrate that a decrease in transcription contributes to the reduction in mRNA levels of nuclear hormone receptors and coactivators. Thus, our results suggest that decreased expression of nuclear hormone receptors RXRα, PPARα, PPARγ, and LXRα, as well as coactivators PGC-1α, PGC-1β, and SRC-1 may contribute to the cytokine induced alterations in hepatic lipid metabolism during the APR.

Abstract:A recent outbreak of Zika virus in Brazil has led to a simultaneous increase in reports of neonatal microcephaly. Zika targets cerebral neural precursors, a cell population essential for cortical development, but the cause of this neurotropism remains obscure. Here we report that the neural RNA-binding protein Musashi-1 (MSI1) interacts with the Zika genome and enables viral replication. Zika infection disrupts the binding of MSI1 to its endogenous targets, thereby deregulating expression of factors implicated in neural stem cell function. We further show that MSI1 is highly expressed in neural progenitors of the human embryonic brain, and is mutated in individuals with autosomal recessive primary microcephaly. Selective MSI1 expression in neural precursors could therefore explain the exceptional vulnerability of these cells to Zika infection. Main text:Zika virus (ZIKV) recently emerged as a major public health risk because of its devastating effect on fetal neurodevelopment (1-3). ZIKV was first isolated in Uganda in 1947, and the virus subsequently spread through Asia, and from there to the Americas (4). A causal link between ZIKV infection and congenital brain malformations became apparent in 2016 following an outbreak in Brazil (1). Brazilian ZIKV is closely related to the Asian-lineage strain, which affected New Caledonia and French Polynesia, where cases of microcephaly were reported retrospectively (5).Intrauterine infections can impair neurodevelopment (6), but ZIKV is highly neurotropic and interferes specifically with fetal brain development causing microcephaly, cortical malformations and intracranial calcifications (7-10). We hypothesized that the singlestranded RNA flavivirus ZIKV may hijack RNA-binding factors present in the developing central nervous system (11). Host RNA-binding proteins are known to interact with untranslated regions (UTRs) to regulate replication, translation and stabilization of viral genomes (11). In silico analysis of the genomic RNA of the Brazilian ZIKV strain, PE243, revealed three consensus binding sites in the 3'UTR for the highly conserved Musashi family of RNA binding proteins, Musashi-1 (MSI1) and Musashi-2 (MSI2), both important translational regulators in stem cells (12)(13)(14)(15). Two sites were conserved between PE243 and the Ugandan MR766 strains (Sites 1, 2), whereas the third (Site 3) was found only in the Asian-lineage strains including PE243 ( Fig. 1A; Fig. S1A, B). By mapping these sites onto a predicted secondary structure of ZIKV 3'UTR, we found all the three to be present on stemloop structures, which are considered optimal for MSI binding (16,17). Moreover, a recent study revealed nucleotide substitutions proximal to Sites 1 and 2 in the Asian-lineage strains, which could indicate positive selection for MSI1 binding during ZIKV evolution (18).To address if the Musashi proteins interacted with ZIKV, we first tested their binding to ZIKV 3'UTR. RNA pull-downs identified binding of MSI1, but not MSI2, to the 3'UTR of PE243 (Fig. 1B) (15). Mutat...

The ErbB3-binding protein 1 (Ebp1) is an important regulator of transcription, affecting eukaryotic cell growth, proliferation, differentiation and survival. Ebp1 can also affect translation and cooperates with the polypyrimidine tract-binding protein (PTB) to stimulate the activity of the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). We report here the crystal structure of murine Ebp1 (p48 isoform), providing the first glimpse of the architecture of this versatile regulator. The structure reveals a core domain that is homologous to methionine aminopeptidases, coupled to a C-terminal extension that contains important motifs for binding proteins and RNA. It sheds new light on the conformational differences between the p42 and p48 isoforms of Ebp1, the disposition of the key protein-interacting motif ( 354 LKALL 358 ) and the RNA-binding activity of Ebp1. We show that the primary RNA-binding site is formed by a Lys-rich motif in the C terminus and mediates the interaction with the FMDV IRES. We also demonstrate a specific functional requirement for Ebp1 in FMDV IRES-directed translation that is independent of a direct interaction with PTB.

The 3C protease (3C pro ) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C pro , a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a ␤-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This ␤-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the ␤-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C pro with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the ␤-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C pro inhibitors.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5′ end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.

Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G‐interacting domain is structurally distinct, although it contains an apical eIF4G‐interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract‐binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin.