Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation (original) (raw)

The different pathways of HIV genomic RNA translation

Biochemical Society Transactions, 2010

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.

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.

A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3

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.

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

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.

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 Leader of Human Immunodeficiency Virus Type 1 Genomic RNA Harbors an Internal Ribosome Entry Segment That Is Active during the G2/M Phase of the Cell Cycle

Journal of Virology, 2003

The 5 leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA contains highly structured domains involved in key steps of the viral life cycle. These RNA domains inhibit cap-dependent protein synthesis. Here we report that the HIV-1 5 leader harbors an internal ribosome entry site (IRES) capable of driving protein synthesis during the G 2 /M cell cycle phase in which cap-dependent initiation is inhibited. The HIV-1 IRES was delineated with bicistronic mRNAs in in vitro and ex vivo assays. The HIV-1 leader IRES spans nucleotides 104 to 336 and partially overlaps the major determinants of genomic RNA packaging. These data strongly suggest that, as for HIV-1 transcription, IRES-mediated translation initiation could play an important role in virus replication during virus-induced G 2 /M cell cycle arrest.

Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV1

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.

Translation initiation of the HIV-1 mRNA

Translation, 2014

Translation initiation of the full-length mRNA of the human immunodeficiency virus can occur via several different mechanisms to maintain production of viral structural proteins throughout the replication cycle. HIV-1 viral protein synthesis can occur by the use of both a capdependant and IRES-driven mechanism depending on the physiological conditions of the cell and the status of the ongoing infection. For both of these mechanisms there is a need for several viral and cellular co-factors for optimal translation of the viral mRNA. In this review we will describe the mechanism used by the full-length mRNA to initiate translation highlighting the role of co-factors within this process. A particular emphasis will be given to the role of the DDX3 RNA helicase in HIV-1 mRNA translation initiation.