Abstract
The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.
Highlights
The genome of retroviruses is formed by two identical RNA molecules that are non-covalently linked [1, 2]
In vitro studies with bovine leukemia virus and HIV-2 RNA indicated that dimerization occurs independently of the downstream dimer linkage structure (DLS) [9, 20], an observation that has been supported by studies with infectious HIV-1 virus particles [14, 21]
We will first compare the nested set of transcripts 1/124, 1/379, and 1/444 for their dimerization ability. These mutant transcripts contain one or more of the motifs that have previously been suggested to be responsible for HIV-2 dimerization, namely the transactivation region (TAR) hairpin (1/124), the primer binding site (PBS) region (1/379), and the dimer initiation site (DIS) hairpin (1/444)
Summary
DIS mutagenesis primersa 444 DIS S mutant 444 DIS SIVsyk 444 DIS HIV-1B 444 DIS HIV-1A/SIVcpz 444 DIS SIVmnd 444 DIS D mutant 444 DIS UP mutant a The mutant DIS palindrome sequence is underlined. CGC TCC ACA CGC TGC CTT TCC TAC CTC GG CGC TCC ACA CGC TGC CTT TTG TAC ATC GG CGC TCC ACA CGC TGC CTT TGC GCG CTC GG CGC TCC ACA CGC TGC CTT TGT GCT CTC GG CGC TCC ACA CGC TGC CTT TGT TAA CTC GG CGC TCC ACA CGC TGC CTT TGC TCG G CGC TCC ACA CGC TGC CTT TGT CGA CTC GG some studies [14, 28] These results indicate the importance of additional sequence motifs in the viral genome that are essential for in vivo RNA dimerization. RNA structure probing and phylogenetic analysis of the leader RNA sequence in different viral isolates suggests that the DIS hairpin exists in an extended form with a stem region of 12 base pairs These results indicate that a very similar RNA dimerization mechanism is operating in all members of the HIV-SIV immunodeficiency viruses
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