The different pathways of HIV genomic RNA translation (original) (raw)
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Lentiviral RNAs can use different mechanisms for translation initiation
Biochemical Society Transactions, 2008
The full-length genomic RNA of lentiviruses can be translated to produce proteins and incorporated as genomic RNA in the viral particle. Interestingly, both functions are driven by the genomic 5′-UTR (5′-untranslated region), which harbours structural RNA motifs for the replication cycle of the virus. Recent work has shown that this RNA architecture also functions as an IRES (internal ribosome entry site) in HIV-1 and -2, and in SIV (simian immunodeficiency virus). In addition, the IRES extends to the gag coding region for all these viruses and this leads to the synthesis of shorter isoforms of the Gag polyprotein from downstream initiation codons. In the present study, we have investigated how different members of the lentivirus family (namely HIV-1 and -2, and SIV) can initiate protein synthesis by distinct mechanisms. For this, we have used the competitive reticulocyte lysate that we have recently described. Our results show that HIV-1 is able to drive the synthesis of the Gag po...
HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon
Nature Structural & Molecular Biology, 2005
Eukaryotic translation initiation begins with assembly of a 48S ribosomal complex at the 5¢ cap structure or at an internal ribosomal entry segment (IRES). In both cases, ribosomal positioning at the AUG codon requires a 5¢ untranslated region upstream from the initiation site. Here, we report that translation of the genomic RNA of human immunodeficiency virus type 2 takes place by attachment of the 48S ribosomal preinitiation complex to the coding region, with no need for an upstream 5¢ untranslated RNA sequence. This unusual mechanism is mediated by an RNA sequence that has features of an IRES with the unique ability to recruit ribosomes upstream from its core domain. A combination of translation assays and structural studies reveal that sequences located 50 nucleotides downstream of the AUG codon are crucial for IRES activity.
The FEBS Journal, 2016
The 5′leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/ tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work we examine the internal ribosome entry site (IRES) located in the 5′leader that drives translation initiation of the viral Gag protein under conditions that hinder capdependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5′leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis.
Viral IRES RNA structures and ribosome interactions
Trends in Biochemical Sciences, 2008
In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES-ribosome complexes are revealing the structural basis of viral IRES' 'hijacking' of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes. Cap-dependent and cap-independent translation in eukaryotes Eukaryotic translation initiation is a tightly regulated process. The canonical mechanism begins with the recognition of the 5′ modified nucleotide cap on the mRNA, it requires many initiation factor (eIF) proteins, uses ribosome scanning (movement of the ribosomal subunit in a 5′ to 3′ direction along the mRNA) to find the start codon and culminates with an 80S ribosome located on the mRNA, ready to begin protein synthesis (reviewed recently in [1]). The 80S ribosome contains a large (50S in bacteria, 60S in eukaryotes) and a small (30S in bacteria, 40S in eukaryotes) ribosomal subunit (Figure 1). During translation on the 80S ribosome, the RNA moves through the decoding groove on the small subunit where it is 'read' by tRNAs that move through the space between the two subunits. There are three tRNA-binding sites on the ribosome, designated as the aminoacyl-site (A-site), the peptidyl-site (P-site) and the exit-site (E-site) (Figure 1). In canonical translation initiation, the initiator tRNA is in the P-site and addition of a tRNA to the A-site leads to peptide-bond formation and translocation. Overall, it is important to remember that the canonical pathway of translation initiation begins when the 5′ cap is recognized. Although the canonical cap-and scanning-dependent mechanism accounts for protein synthesis on the majority of eukaryotic mRNAs, it cannot account for translation initiation on all messages, including certain viral RNAs. For example, poliovirus (PV) is a single-stranded positive-sense RNA virus with no 5′-cap but with a genome-linked peptide (VPg) on its 5′ end, hence, the canonical signal for ribosome recruitment is not present. The mystery of precisely
PLOS One, 2012
The 59untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 59UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 59UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 59UTR of some of the HIV-1 variants was enhanced relative to that observed for the 59UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 59UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-59UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.
Nucleic Acids …, 2011
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.
Nucleic Acids Research, 2011
Translation initiation on HIV genomic RNA relies on both cap and Internal Ribosome Entry Site (IRES) dependant mechanisms that are regulated throughout the cell cycle. During a unique phenomenon, the virus recruits initiation complexes through RNA structures located within Gag coding sequence, downstream of the initiation codon. We analyzed initiation complexes paused on the HIV-2 gag IRES and revealed that they contain all the canonical initiation factors except eIF4E and eIF1. We report that eIF3 and the small ribosomal subunit bind HIV RNA within gag open reading frame. We thus propose a novel two step model whereby the initial event is the formation of a ternary eIF3/40S/IRES complex. In a second step, dependent on most of the canonical initiation factors, the complex is rearranged to transfer the ribosome on the initiation codons. The absolute requirement of this large structure for HIV translation defines a new function for a coding region. Moreover, the level of information compaction within this viral genome reveals an additional level of evolutionary constraint on the coding sequence. The conservation of this IRES and its properties in rapidly evolving viruses suggest an important role in the virus life cycle and highlight an attractive new therapeutic target.
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. Citation: Monette A, Valiente-Echeverría F, Rivero M, Cohen ÉA, Lopez-Lastra M, et al. (2013) Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis. PLoS ONE 8(7): e68108.