Cancer immunotherapy seeks to boost the host’s immune system to respond to tumor antigens. The adaptive immune system comprises of two arms, one that elicits a cellular immune response and one that elicits a humoral immune response. Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides presented to them in the context of class I major histocompatibility complex (MHC) molecules and are capable of killing tumor cells. CTL are educated to discriminate between foreign and self-antigen. Tumors frequently express self-antigen which usually makes them poorly immunogenic. Because tumors are genetically unstable, they may present excess self peptides and/or peptides in a reading frame different from wild type self proteins. These frameshift (FS) peptides, are caused by an insertion or deletion of nucleotides that disrupt translation of the normal reading frame and alters the protein produced such that it is non-self. Binding affinity, dissociation rate and the overall stability of the peptide/MHC/β₂-microglobulin complex are important considerations in determining the immunogenicity of a given peptide. Interaction between the anchor residues in a peptide and binding pockets in MHC are essential, but this interaction is not always strong enough to stimulate T cell responses. This indicates that not all amino acids of the peptide ligand bound to MHC are equally important for the functional outcome of the receptor engagement and that other amino acid residues in the sequence are important for binding. Optimized peptide ligands (OPL) are analogues derived from natural wild type antigenic peptides that contain amino acid substitutions at anchor and auxiliary residues. OPL can be rationally designed to generate a more robust immune response compared to that of the wild-type peptide. Active immunotherapy using OPL of tumor antigen epitopes are designed to elicit tumor-specific CTL that can overcome tolerance and either re-awaken or elicit new T-cell responses to an antigen. The work and principles presented here using brain tumor-derived peptides demonstrates that HLA-A*0201-restricted CTL generated against wild type, frameshift and OPL peptides elicit CTL that were able to recognize and respond to wild type, tumorderived peptides. The response was donor dependent in that not all individuals responded more strongly to OPL; a minority responded better to wild type peptide. This data further suggests that the rational design and testing of multiple peptides for the same epitope should elicit a broader response among different individuals than single peptide immunization.

No description available.

Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack. In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools. Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods. Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.

In evolution alternative genetic trajectories can potentially lead to similar phenotypic outcomes. However, certain trajectories are preferred over others. These preferences bias the genomes of living organisms and the underlying processes can be observed in ongoing evolution. We have studied a variety of biases that can be found in bacterial chromosomes and determined the selective causes and functional consequences for the cell. We have quantified codon usage bias in highly expressed genes and shown that it is selected to optimise translational speed. We further demonstrated that the resulting differences in decoding speed can be used to regulate gene expression, and that the use of ‘non-optimal’ codons can be detrimental to reading frame maintenance. Biased gene location on the chromosome favours recombination between genes within gene families and leads to co-evolution. We have shown that such recombinational events can protect these gene families from inactivation by mobile genetic elements, and that chromosome organization can be selectively maintained because inversions can lead to the formation of unstable hybrid operons. We have used the development of antibiotic resistance to study how different bacterial lifestyles influence evolutionary trajectories. For this we used two distinct pairs of antibiotics and disease-causing bacteria, namely (i) Mycobacterium tuberculosis that is treated with rifampicin and (ii) Escherichia coli that is treated with ciprofloxacin. We have shown that in the slow-growing Mycobacterium tuberculosis, resistance mutations are selected for high-level resistance. Fitness is initially less important, and over time fitness costs can be ameliorated by compensatory mutations. The need for rapid growth causes the selection of ciprofloxacin resistance in Escherichia coli not only to be selected on the basis of high-level resistance but also on high fitness. Compensatory evolution is therefore not required and is not observed. Taken together, our results show that the evolution of a phenotype is the product of multiple steps and that many factors influence which trajectory is the most likely to occur and be most beneficial. Over time, selection will favour this particular trajectory and lead to biased evolution, affecting genome sequence and organization.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

La terminaison de la traduction intervient lorsqu'un ribosome rencontre un codon stop. Ce dernier est alors reconnu par le facteur eRF1p qui, en interaction avec eRF3p, provoque l'arrêt de la synthèse protéique. L'efficacité de terminaison de la traduction est déterminée par le contexte nucléotidique du codon stop. Ainsi, le contexte peut reprogrammer le codon stop en permettant aux ribosomes d'incorporer un ARNt avec une forte efficacité, on parle alors de translecture. J'ai démontré que chez S. cerevisiae les six nucléotides en 3' du codon stop sont déterminants dans l'efficacité de translecture, et que le motif général -CA(A/G)N(T/C/G)A- permet d'obtenir les plus fortes efficacités de passage des codons stop. Mon travail met en évidence que la translecture, identifiée jusqu'à présent uniquement dans des gènes viraux, est aussi retrouvée dans des gènes nucléaires. Elle permet de modifier l'expression des protéines concernées, en fonction des conditions environnementales. C'est notamment le cas du gène PDE2, pour lequel j'ai montré que la translecture permet de contrôler la stabilité de la phosphodiestérase de l'AMPc, et module ainsi le niveau d'AMPc intracellulaire. L'augmentation du niveau d'AMPc observée dans une souche [PSI+] pourrait permettre d'expliquer la grande diversité des phénotypes associés au facteur [PSI+]. Durant ce travail d'autre gènes soumis à un mécanisme de translecture ont été identifiés : il s'agit des gènes IMP3 et BSC4. Pour certains des autres candidats identifiés par notre approche "sans apriori", l'efficacité de translecture est indépendante de la concentration en facteur eRF3p. Ce résultat pourrait signifier qu'au niveau de ces codons stop, le mécanisme de terminaison est indépendant du facteur eRF3p. Le crible d'une banque multicopie d'ADNg m'a permis d'identifier de nouveaux candidats intervenant dans la terminaison de la traduction, ces résultats suggèrent une relation fonctionelle importante entre le cytosquelette et la terminaison de la traduction. L'étude des modifications des ARNt à mis en évidence le rôle important de la pseudouridination aux positions 38 et 39 dans les mécanismes de recodage (translecture et décalage du cadre de lecture). Ce travail a donc mis en évidence que la terminaison de la traduction est un point de contrôle de l'expression de certains gènes chez S. cerevisiae.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

The repertoire of cellular functions mediated by Ribonucleic acid (RNA) molecules have expanded considerably during the last two decades. The role played by RNA in controlling and regulating gene expression in viruses, prokaryotes and eukaryotes has been a matter of continuous investigations. This interest has arisen primarily due to the discoveries of cisacting RNA structures like riboswitches, ribosensors and frameshift elements, which are found in either the 5’-, 3’-untranslated regions of mRNA or in the open reading frames. These structures control gene expression at the level of translation by either sequestering the Shine-Dalgarno (SD) sequence to regulate translation initiation or modulating ribosomal positions during an active translation process. Very often, these structures comprise of an RNA pseudoknot and it has been observed that these pseudoknots exist in a dynamic equilibrium with other intermediate structures. This equilibrium could be shifted by several factors including presence of ions, metabolites, temperature and external force. RNA pseudoknots represent the most versatile and ubiquitous class of RNA structures in the cell, whose unique folding topology could be exploited in a number of ways by the cellular machinery. In this thesis, a thorough study of programmed -1 ribosomal frameshifting (-1 PRF) process, which is a well known gene regulation event employed by many RNA viruses, was carried out. -1 PRF is a translation recoding process, necessary for viruses to main-tain a stoichiometric ratio of structural: enzymatic proteins. This ratio varies among different viral species. At the heart of this process, lies an RNA pseudoknot accompanied by a seven nucleotide long sequence motif, which pauses an actively translating ribosome on mRNA and causes it to shift its reading frame. The frameshift inducing efficiency of pseudoknot depends on multiple factors, for example the time scale of ribosomal pause and RNA unfolding, subsequent refolding of structure to native/intermediate states and/or environment conditions. With the aim of illustrating the fundamentals of the process, multiple factors involved in -1 PRF were studied. Chapters 2-4 represent distinct aspects of -1 PRF process, while Chapter 5 discusses a different work concerned with nucleocytoplasmic transport of tRNA carried out by nuclear export receptor Exporting. Chapter 1 gives an overview of the different regulatory activities with which RNA structures and sequences are found to be associated and the evolution of these stud-ies. It discusses the different types of structural motifs found to constitute tertiary RNA structure and secondary structure prediction and determination techniques. A brief description of ab initio RNA structure modeling and other relevant tools and methodologies used in this work has been presented. Details of techniques used in each study have been provided in relevant chapters. Chapter 2 describes how local factors like ionic conditions, hydration patterns, presence of protonated residues and single residue mutations affect the structural dynamics of an RNA pseudoknot involved in -1 PRF from a plant luteovirus. Single residue mutations in the loop regions or certain base-pair inversions in the stem regions of pseudoknot increase the frameshift inducing ability of the pseudoknot structure, while some others decrease this efficiency. However, it was not clear how the changes made to the wild-type (WT) RNA pseudoknot from Beet Western Yellow Mosaic virus were affecting the global structure in terms of its dynamics and other parameters. To study this, multiple all-atom molecular dynamics simulations (MD) were performed on WT and mutant structures created in silico. The effect of presence and absence of magnesium ions on the structural geometry was also studied. The analysis was done to identify the increase/decrease in the number of hydrogen bonds formed by Watson-Crick base-pairs in stem region or non Watson-Crick pairs between stem and loop. Ionic and water densities were analyzed and the role of potential ribosome-pseudoknot interaction was elaborated. With the aim of mimicking ribosome induced unfolding of an RNA pseudoknot, steered molecular dynamics pulling experiments were performed. This work was done primarily to understand the unfolding pathway of Hairpin(H)-type pseudoknots in general and the intermediate structures formed. Chapter 3 describes the thermodynamics and mechanics associated with the mechanical pulling of -1 PRF inducing RNA pseudoknot and its mutants described in previous chapter. Analysis of the trajectories reveal relative unfolding patterns in terms of disruption of various hydrogen bonds. This study allowed us to pinpoint the kind of intermediate structures being formed during pulling and whether these intermediate structures correspond to any known secondary structures, such as simple stem-loops. This information could be used for gaining insights into the folding pathways of these structures. An RNA pseudoknot stimulates -1 PRF in conjunction with a heptanucleotide “slippery site” and an intervening spacer sequence. A comprehensive study of analyzing the sequence signatures and composition of all overlapping gene segments harboring these frameshift elements from four different RNA virus families was carried out. Chapter 4 describes the sequence composition of all overlapping gene segments in Astroviridae, Coronaviridae, Retroviridae and Luteoviridae viral families which are known to employ -1 PRF process for maintaining their protein products. Sequence analysis revealed preference for GC bases in the structure forming sequence regions. A comparative study between multiple sequence alignment and secondary structure prediction revealed that while pseudoknots have a clear preference for specific base-pairs in their stem regions, viral families that employ a hairpin loop as -1 PRF structure, doesn’t show this preference. Information derived from secondary structure prediction was then used for RNA ab initio modeling to generate tertiary structures. Furthermore, the structural parameters were calculated for the helices of the frameshift inducing pseudoknots and were compared with the values calculated for a set of non -1 PRF inducing H-type pseudo-knots. This study highlighted the differences between -1 PRF pseudoknots and other H-type pseudoknot structures as well as specific sequence and structural preferences of the former. Chapter 5 discusses the dynamics of a tRNA transport factor Exportint (Xpot), which transports mature tRNA molecules from nucleus to cytoplasm and belongs to Importitβ family of proteins. The global conformational dynamics of other transport receptors has been reported earlier, using coarse-grained modeling and Elastic Network Models (ENMs), but a detailed description of the dynamics at an all-atomic resolution was lacking. This transport requires association of Xpot with RanGTP, a G-protein, in the nucleus and hydrolysis of RanGTP in the cytoplasm. The chain of events leading to tRNA release from Xpot after RanGTP hydrolysis was not studied previously. With these objectives, several molecular complexes containing Xpot bound to Ran or tRNA or both in the GTP and GDP ligand states as well as free Xpot structures in nuclear and cytosolic forms were studied. A combination of conventional and accelerated molecular dynamics simulations was used to study these molecular complexes. The study highlighted various aspects associated with tRNA release and conformational change which occurs in Xpot in cytosolic form. The nuclear to cytosolic state transition in Xpot could be attributed to large fluctuations in C-terminal region and dynamic hinge-points located between specific HEAT repeats. A secondary role of Xpot in controlling the quality of tRNA transport has been proposed based on multiple sequence and structure alignment with Importin-β protein. The loss of critical contacts like hydrogen bonds and salt bridges between Xpot/Ran and Xpot/tRNA interface was evaluated in order to study the initial effects of RanGTP hydrolysis and how it influences receptor-cargo binding. This study revealed various aspects of tRNA transport process by Xpot, not understood previously. The results presented in this thesis illustrate the role of RNA sequence elements and pseudoknots present in RNA viruses in modulating -1 PRF process and how multiple environmental factors affect -1 PRF inducing ability of the structure. From the studies of Xpot and its complexes, the effects of GTP hydrolysis leading to tRNA dissociation have been presented and the progression of conformational transition in Xpot after tRNA dissociation has been highlighted. Chapter 6 summarizes major conclusions of this thesis work. The refolding of single stranded RNA chains, subjected to a previous unfolding simulation is studied. Appendix A describes this work and initial results. Appendix B describes the effect of improved molecular dynamics force fields, containing corrections for χ torsion angle for RNA, on the conformation of tertiary RNA structures. Part of the work presented in this thesis has been reported in the following publications. 1.Asmita Gupta and Manju Bansal. Local Structural and Environmental Factors De-fine the Efficiency of an RNA Pseudoknot Involved in Programmed Ribosomal Frameshift Process. J. Phys. Chem. B. 118 (41), pp 11905-11920. 2014 2.Asmita Gupta, Senthilkumar Kailasam and Manju Bansal. Insights Into Nucleo-cytoplasmic Transport of tRNA by Exportin-t. Manuscript under review. List of manuscripts that are being prepared from the work reported in Chapter 3 in this thesis. 1 Asmita Gupta and Manju Bansal. The role of sequence effects on altering the un-folding pathway of an RNA pseudoknot: a steered molecular dynamics study. Manuscript in preparation. 2 Asmita Gupta and Manju Bansal. Molecular basis for nucleocytoplasmic transport of tRNA by Exportin-t. Journal of Biomolecular Structure and Dynamics, May;33 Suppl 1:59-60, 2015

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable du syndrome de l’immunodéficience acquise (SIDA). Il faut identifier de nouvelles cibles pour le développement d’agents anti-VIH-1, car ce virus développe une résistance aux agents présentement utilisés. Notre but est d’approfondir la caractérisation de l’étape du changement de cadre de lecture ribosomique en -1 (déphasage -1) nécessaire à la production du précurseur des enzymes du VIH-1. Ce déphasage est programmé et effectué par une minorité de ribosomes lorsqu’ils traduisent la séquence dite glissante à un endroit spécifique de l’ARN messager (ARNm) pleine-longueur du VIH-1. L’efficacité de déphasage est contrôlée par le signal stimulateur de déphasage (SSF), une tige-boucle irrégulière située en aval de la séquence glissante. La structure du SSF est déroulée lors du passage d’un ribosome, mais elle peut se reformer ensuite. Nous avons montré que des variations de l’initiation de la traduction affectent l’efficacité de déphasage. Nous avons utilisé, dans des cellules Jurkat-T et HEK 293T, un rapporteur bicistronique où les gènes codant pour les luciférases de la Renilla (Rluc) et de la luciole (Fluc) sont séparés par la région de déphasage du VIH-1. La Rluc est produite par tous les ribosomes traduisant l’ARNm rapporteur alors que la Fluc est produite uniquement par les ribosomes effectuant un déphasage. L’initiation de ce rapporteur est coiffe-dépendante, comme pour la majorité des ARNm cellulaires. Nous avons examiné l’effet de trois inhibiteurs de l’initiation et montré que leur présence augmente l’efficacité de déphasage. Nous avons ensuite étudié l’effet de la tige-boucle TAR, qui est présente à l’extrémité 5’ de tous les ARNm du VIH-1. TAR empêche la liaison de la petite sous-unité du ribosome (40S) à l’ARNm et module aussi l’activité de la protéine kinase dépendante de l’ARN double-brin (PKR). L’activation de PKR inhibe l’initiation en phosphorylant le facteur d’initiation eucaryote 2 (eIF2) alors que l’inhibition de PKR a l’effet inverse. Nous avons étudié l’effet de TAR sur la traduction et le déphasage via son effet sur PKR en utilisant TAR en trans ou en cis, mais à une certaine distance de l’extrémité 5’ afin d’éviter l’interférence avec la liaison de la 40S. Nous avons observé qu’une faible concentration de TAR, qui active PKR, augmente l’efficacité de déphasage alors qu’une concentration élevée de TAR, qui inhibe PKR, diminue cette efficacité. Nous avons proposé un modèle où des variations de l’initiation affectent l’efficacité de déphasage en modifiant la distance entre les ribosomes parcourant l’ARNm et, donc, la probabilité qu’ils rencontrent un SSF structuré. Par la suite, nous avons déterminé l’effet de la région 5’ non traduite (UTR) de l’ARNm pleine-longueur du VIH-1 sur l’efficacité de déphasage. Cette 5’UTR contient plusieurs régions structurées, dont TAR à l’extrémité 5’, qui peut interférer avec l’initiation. Cet ARNm a une coiffe permettant une initiation coiffe-dépendante ainsi qu’un site d’entrée interne des ribosomes (IRES), permettant une initiation IRES-dépendante. Nous avons introduit cette 5’UTR, complète ou en partie, comme 5’UTR de notre ARNm rapporteur bicistronique. Nos résultats démontrent que cette 5’UTR complète inhibe l’initiation coiffe dépendante et augmente l’efficacité de déphasage et que ces effets sont dus à la présence de TAR suivie de la tige-boucle Poly(A). Nous avons aussi construit un rapporteur tricistronique où les ribosomes exprimant les luciférases utilisent obligatoirement l’IRES. Nous avons observé que cette initiation par l’IRES est faible et que l’efficacité de déphasage correspondante est également faible. Nous avons formulé une hypothèse pour expliquer cette situation. Nous avons également observé que lorsque les deux modes d’initiation sont disponibles, l’initiation coiffe dépendante est prédominante. Finalement, nous avons étudié l’effet de la protéine virale Tat sur l’initiation de la traduction et sur l’efficacité de déphasage. Nous avons montré qu’elle augmente l’initiation de la traduction et que son effet est plus prononcé lorsque TAR est située à l’extrémité 5’ des ARNm. Nous proposons un modèle expliquant les effets de Tat sur l’initiation de la traduction par l’inhibition de PKR ainsi que par des changements de l’expression de protéines cellulaires déroulant TAR. Ces résultats permettent de mieux comprendre les mécanismes régissant le déphasage du VIH-1, ce qui est essentiel pour le développement d’agents anti-déphasage. / The human immunodeficiency virus type 1 (HIV-1) is responsible for the acquired immune deficiency syndrome (AIDS). HIV-1 develops a resistance towards the inhibitors used to treat infected patients. It is thus important to identify new targets for the development of novel antiretroviral agents. The aim of our work was to better characterize the programmed -1 ribosomal frameshift which generates the precursor of HIV-1 enzymes. The frameshift occurs at a specific sequence of HIV-1 full-length messenger RNA (mRNA), the slippery sequence, and is performed by a minority of the ribosomes translating this mRNA. The frameshift efficiency is controlled by the frameshift stimulatory signal (FSS), an irregular stem-loop located downstream of the slippery sequence. FSS structure is unfolded by every ribosome translating this region and can refold afterwards. We showed that HIV-1 frameshift efficiency is affected by changes in the rate of translation initiation. We transfected Jurkat-T and HEK 293T cells with a bicistronic reporter that contains the frameshift region of HIV-1 between the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) genes. Rluc is produced by all ribosomes translating this reporter whereas only ribosomes that make a –1 frameshift produce Fluc. The translation of the reporter is initiated via a cap-dependant mode, like the majority of cellular mRNAs. We first determined the effect of three inhibitors of translation initiation. We showed that their presence increases the frameshift efficiency. We next determined the impact of the TAR stem loop, which is located at the 5’end of every HIV-1 mRNA. TAR is known to impair the binding of the small subunit of the ribosome (40S) to the mRNA. TAR also modulates the activity of the double-stranded RNA-dependent protein kinase (PKR). When PKR is activated, it phosphorylates the eukaryotic initiation factor 2 (eIF2), inhibiting translation initiation. The inhibition of PKR has the opposite effect. We studied the effect of TAR on PKR by positioning TAR at a distance of the 5’ end where it cannot interfere with the binding of the 40S. Our results showed that a small amount of TAR, which activates PKR, increases the frameshift efficiency whereas a large amount of TAR, which inhibits PKR, decreases it. A model is presented where the variations of translation initiation modulate HIV-1 frameshift efficiency by altering the distance between the elongating ribosomes. This influences the probability that these ribosomes encounter or not a folded FSS. We next observed the effect of the 5’ untranslated region (UTR) of HIV-1 full length mRNA on its frameshift efficiency. This 5’UTR contains several structured parts, including TAR at the 5’end, which can inhibit translation initiation. This mRNA has a cap and an internal ribosome entry site (IRES) and could then use a cap dependent and an IRES-dependent mode of translation initiation. We replaced the 5’UTR of our bicistronic reporter mRNA by the complete 5’UTR of HIV-1 full-length mRNA or a part of it. Our results showed that the presence of the complete 5’UTR inhibits cap-dependent initiation of translation and increases the frameshift efficiency. Those effects are mostly due to the presence of TAR followed by a Poly(A) stem-loop. We also constructed a tricistronic reporter where the ribosomes translating the luciferases have to use an IRES-dependent initiation mode. The rate of this initiation was low and the frameshift efficiency obtained was also low. We proposed a hypothesis accounting for this situation. We also observed that when both initiation modes are available, the cap-dependent mode seems to be highly favored. Finally, we studied the impact of the Tat viral protein on translation initiation and frameshift efficiency. We showed that the presence of Tat increases translation initiation and decreases the frameshift efficiency. Those effects are more important when TAR is present at the 5’end of mRNA. We propose a model explaining the effects of Tat on translation initiation by the inhibition of PKR and by changes in the expression of cellular proteins that are able to unfold TAR. Our results allow us to better understand the mechanisms controlling HIV-1 frameshift, which will help in the development of drugs targeting the HIV-1 frameshift.