Mechanism of HIV-1 Tat RNA translation and its activation by the Tat protein

Retrovirology Mechanism of HIV-1 Tat RNA translation and its activation by the Tat protein Nicolas Charnay 2 Roland Ivanyi-Nagy 2 Ricardo Soto-Rifo 1 Thophile Ohlmann 1 Marcelo Lpez-Lastra 0 Jean-Luc Darlix 2 0 Laboratorio de Virologia Molecular, Centro de Investigaciones Medicas, Facultad de Medicina, Pontificia Universidad Catolica de Chile , Marcoleta 391, Santiago , Chile 1 TEV, Unite de Virologie Humaine INSERM 758, IFR 128, ENS de Lyon , 46 allee d'Italie, 69364 Lyon , France 2 LaboRetro, Unite de Virologie Humaine INSERM 758, IFR 128, ENS de Lyon , 46 allee d'Italie, 69364 Lyon , France Background: The human immunodeficiency virus type 1 (HIV-1) Tat protein is a major viral transactivator required for HIV-1 replication. In the nucleus Tat greatly stimulates the synthesis of full-length transcripts from the HIV-1 promoter by causing efficient transcriptional elongation. Tat induces elongation by directly interacting with the bulge of the transactivation response (TAR) RNA, a hairpin-loop located at the 5'-end of all nascent viral transcripts, and by recruiting cellular transcriptional co-activators. In the cytoplasm, Tat is thought to act as a translational activator of HIV-1 mRNAs. Thus, Tat plays a central role in the regulation of HIV-1 gene expression both at the level of mRNA and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of Tat can be made early on in HIV-1 infected cells to sustain its own synthesis. To address this issue we studied translation of the Tat mRNA in vitro and in human cells using recombinant monocistronic and dicistronic RNAs containing the 5' untranslated region (5'-UTR) of Tat RNA. Results: This study shows that the Tat mRNA can be efficiently translated both in vitro and in cells. Furthermore, our data suggest that translation initiation from the Tat mRNA probably occurs by a internal ribosome entry site (IRES) mechanism. Finally, we show that Tat protein can strongly stimulate translation from its cognate mRNA in a TAR dependent fashion. Conclusion: These results indicate that Tat mRNA translation is efficient and benefits from a feedback stimulation by the Tat protein. This translational control mechanism would ensure that minute amounts of Tat mRNA are sufficient to generate enough Tat protein required to stimulate HIV-1 replication. - Background The human immunodeficiency virus type 1 (HIV-1) encodes for the three canonical polyprotein precursors Gag, Pol, and Env, which are required for the formation of infectious viral particles by infected cells. In addition, HIV-1 encodes for six regulatory proteins, among which the Tat and the Rev factors are absolutely required for viral gene expression at the transcriptional and post-transcriptional levels in infected cells [1]. HIV-1 Tat is a small basic protein that mainly localizes to the nucleus of infected cells, where it acts as a potent transcriptional activator that is indispensable for the synthesis of the full length viral RNA (reviewed in [2-4]). Transcriptional activation by Tat is mediated by multiple interactions between Tat and the nascent viral TAR RNA and between Tat and cellular factors involved [5] in transcription initiation and elongation such as P-TEFb [4-11]. In addition, Tat has been shown to stimulate translation of viral mRNAs [12-14]. Importantly, this cytoplasmic function of Tat seems to require a nuclear experience, since the RNA-protein complex formed between Tat protein and nuclear factors must be assembled in the nucleus in order to later exert its function in the cytoplasm [12-14]. Thus, the HIV-1 Tat protein plays a central role in the regulation of HIV-1 gene expression both at the level of transcription and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of this viral protein can be made early on in HIV-1 infected cells to sustain its own synthesis. Soon after completion of viral DNA synthesis by reverse transcriptase and before its integration into the host genome, the viral DNA can be transcribed, but this generates only low levels of fully spliced viral mRNAs encoding Tat and Nef [15]. These observations led us to hypothesize that Tat mRNA is translated even under conditions where it is present in minute quantities together with a high concentration of cellular mRNAs. Translation of mRNA into protein represents an essential step in gene expression. The regulation of translation is a mechanism used to modulate gene expression in a wide range of biological situations including cell growth, development and the response to biological cues or environmental stresses such as viral infection [16-20]. During viral infection at least two general modes of translational control can be envisaged. The first represents a global control, in which the translation of most cellular mRNAs is regulated. This is evident during the infection of some members of the Picornaviridae [18-20] where global regulation mainly occurs by the modification of translation initiation factors. The second corresponds to a mRNA-specific control, whereby the translation of a particular mRNA or a defined group of mRNAs is modulated without affecting general protein biosynthesis or the translational status of the cellular transcriptome as a whole. Translational control of a specific mRNA is normally driven by regulatory protein complexes that recognize particular elements that are usually present in the 5' and/ or 3' untranslated regions (UTRs) of the target mRNA [2124]. It is well recognized that translation control of protein synthesis is mostly exerted at the initiation step. Translation initiation of eukaryotic mRNAs mostly occurs by a scanning mechanism, whereby the 40S ribosomal subunit binds to the mRNA 5' cap structure and scans the RNA in the 5' to 3' direction until an initiation codon in a favourable 'Kozak' context is encountered [25]. Translation initiation involves the recognition of the mRNA 5' cap structure by eIF4F, which is composed of eIF4E, which binds the 5' cap, eIF4A, and eIF4G, which links the mRNA 5' cap (via eIF4E)] and the 40S ribosomal subunit (via eIF3) [26,27]. Studies on picornavirus protein synthesis led to the discovery of an alternative mechanism of translation initiation, via an internal ribosome entry segment (IRES) [28-30]. A major difference between cap-dependent versus IRES-mediated ribosome binding and initiation of translation is that the eIF4E component of the eIF4F complex is dispensable for most of the latter activity [31,32]. At present IRESes are defined solely by functional criteria and cannot yet be predicted by the presence of characteristic RNA sequences or structural motifs [30,33]. Despite these apparent experimental restraints, since the initial characterization of IRESes in Picornaviridae, viruses from other families including several members of the Retroviridae were found to initiate translation via (...truncated)