Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsThese 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.
Highlights
The trans-acting responsive (TAR) hairpin is present at both the 5′ and 3′ end of the Human immunodeficiency virus type-1 (HIV-1) RNA genome
We previously designed an HIV-1 variant in which the Tat-TAR transcription mechanism was inactivated through mutation and functionally replaced by the doxycycline-inducible Tet-On gene regulation system (Figure 1A)
These results indicate that the reduced packaging of unspliced RNA and the increased packaging of spliced viral transcripts were due to opening of the 5’ TAR hairpin structure
Summary
The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The first 97 nucleotides (nt) of this leader RNA consist of a repeat region (R) that is 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 transcription activation by binding the viral Tat protein and the cyclin T1 subunit of the positive transcriptional elongation factor (pTEFb) [3,4]. The polyA hairpin masks the polyadenylation signal AAUAAA, and its stability is delicately balanced to prevent premature polyadenylation at
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