Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

Das, Vrolijk, Harwig Alex, Berkhout, Berkhout Ben

Description

The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.

Sujets

• Subtypes of HIV
• Tar
• Dimer
• Packaging
• Nucleic acid structure

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Daset al. Retrovirology2012,9:59 http://www.retrovirology.com/content/9/1/59

R E S E A R C HOpen Access Opening of the TAR hairpin in the HIV1 genome causes aberrant RNA dimerization and packaging * Atze T Das , Martine M Vrolijk, Alex Harwig and Ben Berkhout

Abstract Background:The TAR hairpin is present at both the 5′and 3′end of the HIV1 RNA genome. The 5′element binds the viral Tat protein and is essential for Tatmediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV1 variant that does not depend on this TatTAR axis for transcription. Mutations that open the 5′stemloop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV1 replication cycle is affected by this conformational change of the leader RNA. Results:We demonstrate that opening the 5′TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions:These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals. Keywords:HIV1, TAR, Dimerization, Packaging, RNA structure

Background Human immunodeficiency virus type1 (HIV1) is a retro virus with an RNA genome of approximately 9 kb that contains nine open reading frames and untranslated regions at the 5′and 3′end. The highly conserved leader RNA at the 5′end contains several important regulatory RNA motifs that are involved in both early and late repli cation steps [1,2]. The first 97 nucleotides (nt) of this leader RNA consist of a repeat region (R) that is also present at the 3′end of viral transcripts (Figure 1A). This repeat allows the first strand transfer step during reverse transcription and can fold into two stemloop structures: the transacting responsive (TAR) element and the polyA hairpin. The 5′TAR hairpin has an important role in tran scription activation by binding the viral Tat protein and the cyclin T1 subunit of the positive transcriptional elong ation factor (pTEFb) [3,4]. The polyA hairpin masks the polyadenylation signal AAUAAA, and its stability is deli cately balanced to prevent premature polyadenylation at

* Correspondence: [e-mail] Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands

the 5′end, yet allow efficient polyadenylation at the 3′ end [5,6]. Two important RNA elements involved in the initiation of reverse transcription, the primer binding site (PBS) and the primer activation signal (PAS), are posi tioned downstream of the 5′R region in the untranslated leader (Figure 1A) [7,8]. Additional RNA signals include the dimerization initiation signal (DIS), the major splice donor site (SD) and the packaging signalΨ. The DIS hair pin presents a 6nt palindromic loop sequence for kissing loop base pairing and RNA dimerization. The SD site is used for the production of all spliced transcripts, and the stability of this hairpin modulates the splicing efficiency [9]. TheΨsignal is exclusively present in unspliced tran scripts and is important for packaging of the RNA genome into virions. This packaging signal is still poorly defined, and other cisacting sequences in the HIV1 leader RNA have been suggested to contribute to the packaging efficiency, including the upstream TAR and polyA hair pins [1016]. In vitrostudies demonstrated that the HIV1 leader RNA cannot only fold the branched multiple hairpin (BMH) conformation, but also an alternative conformation in which DIS sequences interact with the polyA region [2022]. This longdistance interaction (LDI) prevents

© 2012 Das et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.