Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors

The 5' leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA harbors an internal ribosome entry site (IRES) that is functional during the G2/M phase of the cell cycle. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. We used 'standard' chemical and enzymatic probes and an 'RNA SHAPE' analysis to model the structure of the HIV-1 5' leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. We also assessed the impact of mutations on IRES function. Strikingly, mutations did not significantly affect IRES activity suggesting that the requirement for pre-formed stable secondary or tertiary structure within the HIV-1 IRES may not be as strict as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5' leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins.     

The 5' UTR of HIV-1 full-length mRNA and the Tat viral protein modulate the programmed -1 ribosomal frameshift that generates HIV-1 enzymes

Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed -1 ribosomal frameshift. Here, using dual-luciferase reporters, we investigated whether the highly structured 5' untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency. We showed that, when the 5' UTR of HIV-1 mRNA occupies the 5' end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold in Jurkat T-cells, compared with a control dual-luciferase reporter with a short unstructured 5' UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5' end of the messenger. HIV-1 mRNA 5' UTR also contains an internal ribosome entry site (IRES), but we showed that, when the cap-dependent initiation mode is available, the IRES is not used or is weakly used. However, when the ribosomes have to use the IRES to translate the dual-luciferase reporter, the frameshift efficiency is comparable to that of the control dual-luciferase reporter. The decrease in cap-dependent initiation and the accompanying increase in frameshift efficiency caused by the 5' UTR of HIV-1 mRNA is antagonized, in a dose-dependent way, by the Tat viral protein. Tat also stimulates the IRES-dependent initiation and decreases the corresponding frameshift efficiency. A model is presented that accounts for the variations in frameshift efficiency depending on the 5' UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication.     

Characterization of an internal ribosomal entry segment in the 5' leader of murine leukemia virus env RNA

The 5' untranslated region, also called the leader, of oncoretroviruses and lentiviruses is long and is formed of several structured domains critically important in virus replication. The 5' leader of murine leukemia virus (MLV) RNA contains an internal ribosomal entry segment (IRES) which promotes synthesis of Gag and glyco-Gag polyprotein precursors. In the present study we investigated the translational features of the 5' leader of MLV subgenomic RNA (env RNA) encoding the Env polyprotein precursor. When the env leader was inserted between two genes, such as lacZ and the neomycin resistance cassette, in a dicistronic vector, it allowed IRES-dependent translation of the 3' cistron in the rabbit reticulocyte lysate system and in murine cells. The drug rapamycin and the foot-and-mouth disease virus L protease, known to inhibit cap-dependent translation, caused an enhancement of the translation driven by the env leader sequence, consistent with an IRES activity promoting Env expression. Analysis of several deletion mutants led us to localize the minimal env IRES between the splice junction and the env AUG start codon.     

Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis

The precursor group-specific antigen (pr55^Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55^Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55^Gag expression, the synthesis of other HIV-1 proteins, including that of pr160^Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55^Gag for virus assembly and production.     

Heterogeneous nuclear ribonucleoprotein C modulates translation of c-myc mRNA in a cell cycle phase-dependent manner

The c-myc proto-oncogene plays a key role in the proliferation, differentiation, apoptosis, and regulation of the cell cycle. Recently, it was demonstrated that the 5' nontranslated region (5' NTR) of human c-myc mRNA contains an internal ribosomal entry site (IRES). In this study, we investigated cellular proteins interacting with the IRES element of c-myc mRNA. Heterogeneous nuclear ribonucleoprotein C (hnRNP C) was identified as a cellular protein that interacts specifically with a heptameric U sequence in the c-myc IRES located between two alternative translation initiation codons CUG and AUG. Moreover, the addition of hnRNP C1 in an in vitro translation system enhanced translation of c-myc mRNA. Interestingly, hnRNP C was partially relocalized from the nucleus, where most of the hnRNP C resides at interphase, to the cytoplasm at the G(2)/M phase of the cell cycle. Coincidently, translation mediated through the c-myc IRES was increased at the G(2)/M phase when cap-dependent translation was partially inhibited. On the other hand, a mutant c-myc mRNA lacking the hnRNP C-binding site, showed a decreased level of translation at the G(2)/M phase compared to that of the wild-type message. Taken together, these findings suggest that hnRNP C, via IRES binding, modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.     

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5' untranslated regions are efficiently translated in cells by a cap-dependent mechanism

The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 5' cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 5' untranslated region (5'UTR), contain extensive secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 5'UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap-dependent mechanism.     

The different pathways of HIV genomic RNA translation

Lentiviruses, the prototype of which is HIV-1, can initiate translation either by the classical cap-dependent mechanism or by internal recruitment of the ribosome through RNA domains called IRESs (internal ribosome entry sites). Depending on the virus considered, the mechanism of IRES-dependent translation differs widely. It can occur by direct binding of the 40S subunit to the mRNA, necessitating a subset or most of the canonical initiation factors and/or ITAF (IRES trans-acting factors). Nonetheless, a common feature of IRESs is that ribosomal recruitment relies, at least in part, on IRES structural determinants. Lentiviral genomic RNAs present an additional level of complexity, as, in addition to the 5'-UTR (untranslated region) IRES, the presence of a new type of IRES, embedded within Gag coding region was described recently. This IRES, conserved in all three lentiviruses examined, presents conserved structural motifs that are crucial for its activity, thus reinforcing the link between RNA structure and function. However, there are still important gaps in our understanding of the molecular mechanism underlying IRES-dependent translation initiation of HIV, including the determination of the initiation factors required, the dynamics of initiation complex assembly and the dynamics of the RNA structure during initiation complex formation. Finally, the ability of HIV genomic RNA to initiate translation through different pathways questions the possible mechanisms of regulation and their correlation to the viral paradigm, i.e. translation versus encapsidation of its genomic RNA.     

In vitro studies reveal that different modes of initiation on HIV-1 mRNA have different levels of requirement for eukaryotic initiation factor 4F

Expression of the two isoforms p55 and p40 of HIV-1 Gag proteins relies on distinct translation initiation mechanisms, a cap-dependent initiation and two internal ribosome entry sites (IRESs). The regulation of these processes is complex and remains poorly understood. This study was focused on the influence of the 5'-UTR and on the requirement for the eukaryotic initiation factor (eIF)4F complex components. By using an in?vitro system, we showed substantial involvement of the 5'-UTR in the control of p55 expression. This highly structured 5'-UTR requires the eIF4F complex, especially RNA helicase eIF4A, which mediates initiation at the authentic AUG codon. In addition, the 5'-UTR regulates expression in an RNA concentration-dependent manner, with a high concentration of RNA triggering specific reduction of full-length Gag p55 production. HIV-1 genomic RNA also has the ability to use a strong IRES element located in the gag coding region. We show that this mechanism is particularly efficient, and that activity of this IRES is only poorly dependent on RNA helicase eIF4A when the viral 5'-UTR is removed. HIV-1 genomic mRNA exhibits in?vitro translational features that allow the expression of Gag p55 protein by different mechanisms that involve different requirements for eIF4E, eIF4G, and eIF4A. This suggests that HIV-1 could adapt to its mode of translation according to the availability of the initiation factors in the infected cell.     

Mechanism of ribosome recruitment by hepatitis C IRES RNA

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.     

An internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic RNA

The retroviral genomic RNA is the messenger for the synthesis of the group-specific antigen (gag) and polymerase precursors of the major structural proteins and enzymes of the virion. The 5'-untranslated leader of the simian immunodeficiency virus (SIV) genomic RNA is formed of highly structured domains involved in key steps of the viral life cycle. Thus, the presence of stable RNA structures between the 5'-cap and the gag start codon are thought to strongly inhibit scanning of a 43 S preinitiation ribosomal complex. This prompted us to look for an alternative to the canonical ribosome scanning. By using a standard bicistronic assay in the rabbit reticulocyte lysate, we show that the SIVmac 5'-leader contains an internal ribosome entry segment (IRES) and that gene expression driven by this IRES is stimulated upon cleavage of eukaryotic initiation factor 4G. Deletion analysis revealed that the sequence between the major splice donor and the gag AUG codon is required for IRES activity. DNA transfection and viral transduction experiments in both NIH-3T3 and COS-7 cells confirmed that translation driven by the SIV leader is IRES-dependent and thus insensitive to the immunosuppressant rapamycin. Identification of an IRES in SIV is of particular interest for the understanding of lentivirus replication and also for the design of novel lentiviral vectors suitable for gene transfer.     

Minimum internal ribosome entry site required for poliovirus infectivity.

Translation initiation by internal ribosome binding is a recently discovered mechanism of eukaryotic viral and cellular protein synthesis in which ribosome subunits interact with the mRNAs at internal sites in the 5' untranslated RNA sequences and not with the 5' methylguanosine cap structure present at the extreme 5' ends of mRNA molecules. Uncapped poliovirus mRNAs harbor internal ribosome entry sites (IRES) in their long and highly structured 5' noncoding regions. Such IRES sequences are required for viral protein synthesis. In this study, a novel poliovirus was isolated whose genomic RNA contains two gross deletions removing approximately 100 nucleotides from the predicted IRES sequences within the 5' noncoding region. The deletions originated from previously in vivo-selected viral revertants displaying non-temperature-sensitive phenotypes. Each revertant had a different predicted stem-loop structure within the 5' noncoding region of their genomic RNAs deleted. The mutant poliovirus (Se1-5NC-delta DG) described in this study contains both stem-loop deletions in a single RNA genome, thereby creating a minimum IRES. Se1-5NC-delta DG exhibited slow growth and a pinpoint plaque phenotype following infection of HeLa cells, delayed onset of protein synthesis in vivo, and defective initiation during in vitro translation of the mutated poliovirus mRNAs. Interestingly, the peak levels of viral RNA synthesis in cells infected with Se1-5NC-delta DG occurred at slightly later times in infection than those achieved by wild-type poliovirus, but these mutant virus RNAs accumulated in the host cells during the late phases of virus infection. UV cross-linking assays with the 5' noncoding regions of wild-type and mutated RNAs were carried out in cytoplasmic extracts from HeLa cells and neuronal cells and in reticulocyte lysates to identify the cellular factors that interact with the putative IRES elements. The cellular proteins that were cross-linked to the minimum IRES may represent factors playing an essential role in internal translation initiation of poliovirus mRNAs.     

A dormant internal ribosome entry site controls translation of feline immunodeficiency virus

The characterization of internal ribosome entry sites (IRESs) in virtually all lentiviruses prompted us to investigate the mechanism used by the feline immunodeficiency virus (FIV) to produce viral proteins. Various in vitro translation assays with mono- and bicistronic constructs revealed that translation of the FIV genomic RNA occurred both by a cap-dependent mechanism and by weak internal entry of the ribosomes. This weak IRES activity was confirmed in feline cells expressing bicistronic RNAs containing the FIV 5' untranslated region (UTR). Surprisingly, infection of feline cells with FIV, but not human immunodeficiency virus type 1, resulted in a great increase in FIV translation. Moreover, a change in the cellular physiological condition provoked by heat stress resulted in the specific stimulation of expression driven by the FIV 5' UTR while cap-dependent initiation was severely repressed. These results reveal the presence of a "dormant" IRES that becomes activated by viral infection and cellular stress.     

Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by AKT activity and enhanced by rapamycin through a p38 MAPK- and ERK-dependent pathway

The macrolide antibiotic rapamycin inhibits the mammalian target of rapamycin protein (mTOR) kinase resulting in the global inhibition of cap-dependent protein synthesis, a blockade in ribosome component biosynthesis, and G1 cell cycle arrest. G1 arrest may occur by inhibiting the protein synthesis of critical factors required for cell cycle progression. Hypersensitivity to mTOR inhibitors has been demonstrated in cells having elevated levels of AKT kinase activity, whereas cells containing quiescent AKT activity are relatively resistant. Our previous data suggest that low AKT activity induces resistance by allowing continued cap-independent protein synthesis of cyclin D1 and c-Myc proteins. In support of this notion, the current study demonstrates that the human cyclin D1 mRNA 5' untranslated region contains an internal ribosome entry site (IRES) and that both this IRES and the c-myc IRES are negatively regulated by AKT activity. Furthermore, we show that cyclin D1 and c-myc IRES function is enhanced following exposure to rapamycin and requires both p38 MAPK and RAF/MEK/ERK signaling, as specific inhibitors of these pathways reduce IRES-mediated translation and protein levels under conditions of quiescent AKT activity. Thus, continued IRES-mediated translation initiation may permit cell cycle progression upon mTOR inactivation in cells in which AKT kinase activity is relatively low.     

Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.     

Cap-independent translation conferred by the 5' leader of tobacco etch virus is eukaryotic initiation factor 4G dependent

The 5' leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.     

mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nucleotide elements permitting selective AIMV CP expression, we tested capped mRNAs containing structured or unstructured 5' leader sequences in addition to an mRNA containing the poliovirus internal ribosome entry site (IRES). Translations were performed with PI-extracts and extracts prepared from mock-infected HeLa cells (MI-extracts). A number of control criteria demonstrated that the HeLa cells were infected by poliovirus and that the extracts were translationally active. The data strongly indicate that translation of RNAs lacking an internal ribosome entry site, including AIMV CP RNA, was severely compromised in PI-extracts, and we find no evidence that the unstructured AIMV CP RNA 5' leader sequence acts in cis to bypass the poliovirus translational control. Nevertheless, cotranslation assays in the MI-extracts demonstrate that mRNAs containing the unstructured AIMV CP RNA 5' untranslated region have a competitive advantage over those containing the rabbit alpha-globin 5' leader. Previous reports of AIMV CP RNA translation in PI-extracts likely describe inefficient expression that can be explained by residual cap-dependent initiation events, where AIMV CP RNA translation is competitive because of a diminished quantitative requirement for initiation factors.     

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.     

Structure and function of the non-coding regions of hepatitis C viral RNA

At the 5' and 3' end of genomic HCV RNA there are two highly conserved, untranslated regions, 5'UTR and 3'UTR. These regions are organized into spatially ordered structures and they play key functions in regulation of processes of the viral life cycle. Most nucleotides of the region located at the 5' side of the coding sequence serve as an internal ribosomal entry site, IRES, which directs cap-independent translation. The RNA fragment present at the 3' end of the genome is required for virus replication and probably contributes to translation of viral proteins. During virus replication its genomic strand is transcribed into a strand of minus polarity, the replicative strand. Its 3' terminus is responsible for initiation of synthesis of descendant genomic strands. This article summarizes our current knowledge on the structure and function of the non-coding regions of hepatitis C genomic RNA, 5'UTR and 3'UTR, and the complementary sequences of the replicative viral strand.