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Internal initiation is a 5'-end-independent mode of translation initiation engaged by many virus- and putatively some cell-encoded templates. Internal initiation is facilitated by specific RNA tertiary folds, called internal ribosomal entry sites (IRESs), in the 5' untranslated region (UTR) of the respective transcripts. In this review we discuss recent structural insight into how established IRESs first capture and then manipulate the eukaryotic translation machinery through non-canonical interactions and by guiding the intrinsic conformational flexibility of the eukaryotic ribosome. Because IRESs operate with reduced complexity and constitute minimal systems of initiation, comparison with canonical initiation may allow common mechanistic principles of the ribosome to be delineated.

Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.

Eukaryotic translation termination results from the complex functional interplay between two eukaryotic release factors, eRF1 and eRF3, and the ribosome, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, using cryo-electron microscopy (cryo-EM) and flexible fitting, we determined the structure of eRF1–eRF3–guanosine 5′-[β,γ-imido]triphosphate (GMPPNP)-bound ribosomal pretermination complex (pre-TC), which corresponds to the initial, pre-GTP hydrolysis stage of factor attachment. Our results show that eukaryotic translation termination involves a network of interactions between the two release factors and the ribosome. Our structure provides mechanistic insight into the coordination between GTP hydrolysis by eRF3 and subsequent peptide release by eRF1.

The gastrointestinal tract represents a major site for human and simian immunodeficiency virus (HIV and SIV) replication and CD4+T-cell depletion. Despite severe depletion of mucosal CD4+T cells, FOXP3+regulatory CD4+T cells (Treg) are highly increased in the gut mucosa of chronically HIV-infected individuals and may contribute to HIV pathogenesis, either by their immunosuppressive function or as a significant target cell population for virus production. Little is known about the susceptibility of mucosal Tregto viral infection and the longitudinal effect of HIV/SIV infection on Tregdynamics. In this study, we determined the level of SIV infection in Tregand nonregulatory CD4+T cells (non-Treg) isolated from the colon of SIV-infected rhesus macaques. The dynamics of mucosal Tregand alterations in the mucosal CD4+T-cell pool were examined longitudinally. Our findings indicate that mucosal Tregwere less susceptible to productive SIV infection than non-Tregand thus were selectively spared from SIV-mediated cell death. In addition to improved survival, local expansion of Tregby SIV-induced proliferation of the mucosal CD4+T-cell pool facilitated the accumulation of mucosal Tregduring the course of infection. High frequency of mucosal Tregin chronic SIV infection was strongly related to a reduction of perforin-expressing cells. In conclusion, this study suggests that mucosal Tregare less affected by productive SIV infection than non-Tregand therefore spared from depletion. Although SIV production is limited in mucosal Treg, Tregaccumulation may indirectly contribute to viral persistence by suppressing antiviral immune responses.

BACKGROUND: Human immunodeficiency virus Type 1 (HIV-1) assays applying nucleic acid testing (NAT) rely on HIV-1 sequence-specific primers and probes. Their hybridization can be limited or abolished by genetic polymorphisms occurring in the target sequence. STUDY DESIGN AND METHODS: Blood donations are routinely tested for HIV-1/2 antibodies and for HIV-1 RNA in our blood transfusion unit. Recently, HIV-1 RNA was undetectable with an established in-house real-time long terminal repeat (LTR) reverse transcriptase–polymerase chain reaction (RT-PCR) in two cases, whereas serologic assays were positive. The reason for this discrepancy was elucidated by sequencing of the NAT target region in the respective single donations. An improved primer was designed and tested on HIV-1 reference panels and blood donations to ensure reliable detection of HIV-1 RNA. RESULTS: Direct sequencing of the target region, isolated from samples of two unrelated HIV-positive blood donors, revealed one and four mismatches in the hybridization domain of the forward primer, respectively. Both viruses belong to HIV-1 Subtype B. LTR RT-PCR with an additional forward primer was suitable for all strains of HIV-1 tested with high sensitivity. CONCLUSIONS: Surveillance of HIV-1 genetic diversity is essentially required to continually evaluate its impact on performance of diagnostic and patient monitoring assays.

Viral internal ribosomal entry sites (IRESs) mediate end-independent translation initiation. There are 4 major structurally-distinct IRES groups: type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis virus), which are dissimilar except for a Yn-Xm-AUG motif at their 3′ borders, type 3 (e.g., hepatitis C virus), and type 4 (dicistroviruses). Type 2–4 IRESs mediate initiation by distinct mechanisms that are nevertheless all based on specific noncanonical interactions with canonical components of the translation apparatus, such as eukaryotic initiation factor (eIF) 4G (type 2), 40S ribosomal subunits (types 3 and 4), and eIF3 (type 3). The mechanism of initiation on type 1 IRESs is unknown. We now report that domain V of type 1 IRESs, which is adjacent to the Yn-Xm-AUG motif, specifically interacts with the central domain of eIF4G. The position and orientation of eIF4G relative to the Yn-Xm-AUG motif is analogous in type 1 and 2 IRESs. eIF4G promotes recruitment of eIF4A to type 1 IRESs, and together, eIF4G and eIF4A induce conformational changes at their 3′ borders. The ability of mutant type 1 IRESs to bind eIF4G/eIF4A correlated with their translational activity. These characteristics parallel the mechanism of initiation on type 2 IRESs, in which the key event is binding of eIF4G to the J–K domain adjacent to the Yn-Xm-AUG motif, which is enhanced by eIF4A. These data suggest that fundamental aspects of the mechanisms of initiation on these unrelated classes of IRESs are similar.

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.

Translation initiation in eukaryotes leads to the assembly of an 80S ribosome containing aminoacylated initiator tRNA (Met-) in the P (peptidyl) site with its anticodon base-paired to the initiation codon of mRNA. It involves at least eleven eukaryotic initiation factors (eIFs). eIF2, GTP, and Met- form a ternary complex, which together with eIF3, eIF1, and eIF1A binds to the 40S subunit to form a 43S complex (Benne and Hershey 1978). After binding to the 5′-end of mRNA, mediated by eIF4A, eIF4B, and eIF4F, the 43S complex scans downstream until it encounters an AUG triplet in a favorable context, where it stops and forms a stable 48S complex with established codon-anticodon base-pairing. eIF1 plays a key role in initiation codon selection (Yoon and Donahue 1992; Pestova et al. 1998; Pestova and Kolupaeva 2002). Finally, a 60S subunit joins the 48S complex but cannot do so directly, presumably because the interface surface of the 40S subunit is occupied by eIF1, eIF1A, eIF2, and eIF3, which must be displaced. The first step in ribosomal subunit joining is hydrolysis of eIF2-bound GTP and release of eIF2-GDP from 48S complexes (Das and Maitra 2002). eIF2 consists of α, β, and γ subunits. The structure of eIF2γ is characteristic of GTP-binding proteins (Schmitt et al. 2002), but eIF2 does not have intrinsic GTPase activity. Hydrolysis of eIF2-bound GTP is induced by eIF5, a GTPase-activating protein (GAP) specific for eIF2, which provides an “arginine finger” for the catalytic center of eIF2γ (Das and Maitra 2002). eIF5 binds directly to eIF2β but induces the GTPase activity of eIF2 only in those eIF2 ternary complexes that are bound to 40S subunits (Raychaudhuri et al. 1985). A few biochemical experiments indicate that eIF5-induced hydrolysis of eIF2-bound GTP may be weakly AUG-dependent (e.g., Raychaudhuri et al. 1985) but they were done in a minimal system containing only 40S subunits, eIF2 ternary complex, and eIF5, in which AUG triplets also stimulated the prior stage, binding of ternary complexes to 40S subunits. Although in vitro data concerning a link between initiation codon recognition and hydrolysis of eIF2-bound GTP are limited, strong genetic data support the importance of a stringent connection between them: Mutations in eIF5 (which increase its GAP activity), in eIF2γ (which increase eIF2 dissociation from Met- even without GTP hydrolysis) and in eIF2β (which increase eIF2's intrinsic GTPase activity) all enhance initiation at non-AUG codons in yeast (Huang et al. 1997). Thus premature hydrolysis of eIF2-bound GTP and release of eIF2 from initiation complexes reduce the fidelity of initiation and should be avoided. It has been widely assumed that eIF5-induced hydrolysis of eIF2-bound GTP and release of eIF2-GDP from 48S complexes lead to release of all other factors (e.g., Das and Maitra 2002). If this were so, hydrolysis of eIF2-bound GTP should logically suffice for subunit joining. However, it is sufficient only for 60S subunits to join minimal 48S complexes formed on AUG triplets, but not to 48S complexes formed on native mRNA with eIF2, eIF3, eIF1, and eIF1A. In this case, eIF5B is also required (Pestova et al. 2000). It has been suggested that eIF5B's role could be to adjust the position of Met- on the 40S subunit as a prerequisite for subunit joining (Roll-Mecak et al. 2001). However, eIF5B's role in subunit joining could also be because, contrary to the common assumption, one or more of eIF1, eIF1A, and eIF3 are not displaced from the 40S subunit after eIF5-induced eIF2-GDP release. Although eIF1, eIF1A, and eIF3 can all bind to 40S subunits independent of the eIF2 ternary complex (Fraser et al. 2004; Maag and Lorsch 2003), contrary to other reports (e.g., Trachsel and Staehelin 1979), in the absence of eIF2 ternary complex, these factors do not protect 40S subunits from joining with 60S subunits (Chaudhuri et al. 1999; Kolupaeva et al. 2005). Therefore, even if eIF1, eIF1A, and eIF3 were not released from 48S complexes after GTP hydrolysis, their presence should not prevent subunit joining. However, we recently found that eIF3 stably binds to 40S subunits in the presence of single-stranded RNA, forming ternary complexes in which RNA is most likely bound in the mRNA-binding cleft of the 40S subunit, and this prevents 40S subunits from joining with 60S subunits even in the absence of eIF2 ternary complex (Kolupaeva et al. 2005). These data suggest that association of eIF3 with 40S subunits in mRNA-containing 48S complexes after release of eIF2-GDP would potentially block subunit joining, and the role of eIF5B could be to displace eIF3 and possibly eIF1 and eIF1A from the 40S subunit after release of eIF2. This could occur in at least two ways. eIF5B could either actively displace these factors, yielding a free 40S subunit/Met- complex before subunit joining, or it could mediate subunit joining on 48S complexes containing eIF3, eIF1, and eIF1A so that these factors dissociate during the actual subunit joining event. We have investigated these questions and report here that eIF5-induced hydrolysis of eIF2-bound GTP in 48S complexes assembled on mRNA led to release of only eIF2, whereas eIF1 and eIF3 remained bound to 40S subunits. eIF5B does not influence factor displacement in the absence of 60S subunits, and eIF3 and eIF1 are released only during the actual subunit joining event mediated by eIF5B. In the absence of eIF1, eIF5-stimulated hydrolysis of eIF2-bound GTP did not depend on establishment of codon-anticodon interaction and occurred at the same rate in 43S and 48S complexes. In the presence of eIF1, GTP hydrolysis in 43S complexes was much slower than in 43S or 48S complexes assembled in the absence of eIF1. Establishment of codon-anticodon interaction in 48S complexes assembled in the presence of eIF1 relieved eIF1's inhibition. eIF1 therefore plays the role of a negative regulator, which inhibits premature GTP hydrolysis and links codon-anticodon base-pairing with hydrolysis of eIF2-bound GTP. Thus, in addition to its role in initiation codon selection at the stage of 48S complex formation, eIF1 also participates in ensuring the fidelity of initiation at the stage of hydrolysis of eIF2-bound GTP.

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.

The universally conserved eukaryotic initiation factor (eIF) 5B, a translational GTPase, is essential for canonical translation initiation. It is also required for initiation facilitated by the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) RNA. eIF5B promotes joining of 60S ribosomal subunits to 40S ribosomal subunits bound by initiator tRNA (Met-tRNAi(Met)). However, the exact molecular mechanism by which eIF5B acts has not been established. Here we present cryo-EM reconstructions of the mammalian 80S-HCV-IRES-Met-tRNAi(Met)-eIF5B-GMPPNP complex. We obtained two substates distinguished by the rotational state of the ribosomal subunits and the configuration of initiator tRNA in the peptidyl (P) site. Accordingly, a combination of conformational changes in the 80S ribosome and in initiator tRNA facilitates binding of the Met-tRNAi(Met) to the 60S P site and redefines the role of eIF5B as a tRNA-reorientation factor.

Human immunodeficiency virus Type 1 (HIV-1) assays applying nucleic acid testing (NAT) rely on HIV-1 sequence-specific primers and probes. Their hybridization can be limited or abolished by genetic polymorphisms occurring in the target sequence.Blood donations are routinely tested for HIV-1/2 antibodies and for HIV-1 RNA in our blood transfusion unit. Recently, HIV-1 RNA was undetectable with an established in-house real-time long terminal repeat (LTR) reverse transcriptase-polymerase chain reaction (RT-PCR) in two cases, whereas serologic assays were positive. The reason for this discrepancy was elucidated by sequencing of the NAT target region in the respective single donations. An improved primer was designed and tested on HIV-1 reference panels and blood donations to ensure reliable detection of HIV-1 RNA.Direct sequencing of the target region, isolated from samples of two unrelated HIV-positive blood donors, revealed one and four mismatches in the hybridization domain of the forward primer, respectively. Both viruses belong to HIV-1 Subtype B. LTR RT-PCR with an additional forward primer was suitable for all strains of HIV-1 tested with high sensitivity.Surveillance of HIV-1 genetic diversity is essentially required to continually evaluate its impact on performance of diagnostic and patient monitoring assays.

The canonical initiation process is the most complex aspect of translation in eukaryotes. It involves the coordinated interactions of at least 11 eukaryotic initiation factors, 40S and 60S ribosomal subunits, mRNA, and aminoacylated initiator tRNA (Met-tRNA(i)(Met)), as well as binding and hydrolysis of GTP and ATP. The factor requirements for many individual steps in this process, including scanning, initiation codon recognition, and ribosomal subunit joining, have until recently been obscure. We established the factor requirements for these steps by reconstituting the initiation process in vitro from individual purified components of the translation apparatus and developed approaches to explain the mechanism of individual steps and the roles of individual factors and to characterize the structure of initiation complexes. Here we describe protocols for the purification of native initiation factors and for expression and purification of active recombinant forms of all single subunit initiation factors, for the reconstitution of the initiation process, and for determination of the position of ribosomal complexes on mRNA by primer extension inhibition ("toe printing"). We also describe protocols for site-directed ultraviolet (UV) cross-linking to determine the interactions of individual nucleotides in mRNA with components of the initiation complex and for directed hydroxyl radical probing to determine the position of initiation factors on the ribosome.

The canonical initiation process is the most complex aspect of translation in eukaryotes. It involves the coordinated interactions of at least 11 eukaryotic initiation factors, 40S and 60S ribosomal subunits, mRNA, and aminoacylated initiator tRNA (Met-tRNA(i)(Met)), as well as binding and hydrolysis of GTP and ATP. The factor requirements for many individual steps in this process, including scanning, initiation codon recognition, and ribosomal subunit joining, have until recently been obscure. We established the factor requirements for these steps by reconstituting the initiation process in vitro from individual purified components of the translation apparatus and developed approaches to explain the mechanism of individual steps and the roles of individual factors and to characterize the structure of initiation complexes. Here we describe protocols for the purification of native initiation factors and for expression and purification of active recombinant forms of all single subunit initiation factors, for the reconstitution of the initiation process, and for determination of the position of ribosomal complexes on mRNA by primer extension inhibition ("toe printing"). We also describe protocols for site-directed ultraviolet (UV) cross-linking to determine the interactions of individual nucleotides in mRNA with components of the initiation complex and for directed hydroxyl radical probing to determine the position of initiation factors on the ribosome.

The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j−, respectively). Although unlike eIF3j+, eIF3j− does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2•GTP•Met-tRNAiMet ternary complex, eIF3j−/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j− nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.

Lipopolysaccharide (LPS) Binding Protein (LBP) is an acute phase protein with the ability to recognize bacterial LPS and transport it to the CD14 molecule or into HDL particles. It is synthesized in hepatocytes and secreted into the blood stream. LBP levels significantly rise during the acute phase response and levels of LBP may be important for an appropriate host reaction to bacterial challenge and for developing the sepsis syndrome. In order to elucidate the mechanisms of LBP regulation we investigated its transcription pattern and performed promoter studies under experimental conditions mimicking an acute phase scenario. In human hepatoma cell lines stimulation with IL-1 beta, IL-6, TNF-alpha and dexamethasone leads to strong transcriptional activation of the LBP gene in a dose- and time-dependent manner. IL-6 alone induces LBP significantly, whereas IL-1 beta mainly increases the IL-6 effect when applied in combination. Our results furthermore show that AP-1 and C/EBP beta are transcription factors involved in the activation of the LBP gene, as revealed by Luciferase reporter gene analysis and electromobility shift assays. Elucidating the mechanism of transcriptional activation of LBP potentially may help in understanding host-pathogen response patterns and mechanisms involved in the acute phase reaction and in the pathophysiology of sepsis.