HIV-2 RNA dimerization is regulated by intramolecular interactions in vitro

Abstract

Genomic RNA dimerization is an essential process in the retroviral replication cycle. In vitro, HIV-2 RNA dimerization is mediated at least in part by direct intermolecular interaction at stem-loop 1 (SL1) within the 59-untranslated leader region (UTR). RNA dimerization is thought to be regulated via alternate presentation and sequestration of dimerization signals by intramolecular base-pairings. One of the proposed regulatory elements is a palindrome sequence (pal) located upstream of SL1. To investigate the role of pal in the regulation of HIV-2 dimerization, we randomized this motif and selected in vitro for dimerization-competent and dimerization-impaired RNAs. Energy minimization folding analysis of these isolated sequences suggests the involvement of pal region in several short-distance intramolecular interactions with other upstream and downstream regions of the UTR. Moreover, the consensus predicted folding patterns indicate the altered presentation of SL1 depending on the interactions of pal with other regions of RNA. The data suggest that pal can act as a positive or negative regulator of SL1-mediated dimerization and that the modulation of base-pairing arrangements that affect RNA dimerization could coordinate multiple signals located within the 59-UTR.

Mechanisms of Human Immunodeficiency Virus Type 2 RNA Packaging: Efficient trans Packaging and Selection of RNA Copackaging Partners

“…The role of the 6-nt palindromic sequence in RNA dimerization has been the subject of controversy; although in vitro experiments have shown that this 6-nt RNA motif is important for RNA dimerization, cell culture-based experiments have demonstrated that destroying the 6-nt palindromic sequence does not affect RNA dimerization (3,4,13,(29)(30)(31)36). In our report, we show that this palindromic sequence is a major element responsible for the copackaged viral RNA selection.…”

“…Similar to HIV-1, HIV-2 also has a proposed stem-loop structure (SL1) at the 5Ј-untranslated region (UTR) with a 6-nt palindrome at the loop location. Results from in vitro RNA dimerization studies have suggested that this 6-nt palindrome is important for the initiation of dimerization (13), although other in vitro studies suggest that another palindromic RNA element (pal) upstream of the 6-nt loop sequence is also important for dimerization (3,29). Furthermore, cell culture experiments indicate that the 6-nt palindrome in SL1 is dispensable for RNA dimerization and virus replication (36); however, mutations in pal can have detrimental effects on RNA dimerization and virus replication (4,30,36).…”

Randomization and In Vivo Selection Reveal a GGRG Motif Essential for Packaging Human Immunodeficiency Virus Type 2 RNA

“…TAR, poly(A) signal, PBS, , SL1, SD, and gag represent the transactivation region, the poly (A) signal domain, the primer binding site, the packaging signal, the stem-loop 1, the major splice donor site, and the 5Ј end of the Gag protein coding region, respectively. (B) The secondary structure of ⌿-SL1 region (nt 380 to 448) (3,15,35). The pal element is a 10-nt palindrome sequence (nt 392 to 401) within ⌿, located immediately upstream of SL1 and is represented by boldface letters.…”

“…1B) (25). Mutations of the pal element revealed its involvement in RNA dimerization both in vitro and in vivo (3,25,26,30).…”

“…Reversion analysis of pal mutant viruses in long-term culture suggested that the 3Ј side of pal (GCUCC-3Ј) interacts with a downstream sequence and forms an extension of SL1 called stem B that is required for viral replication and genomic encapsidation (26). In parallel, an in vitro SELEX study found the 3Ј side of pal to be involved in the stem B structure that regulates SL1-mediated HIV-2 RNA dimerization in vitro (3). Finally, mutations in the pal/SL1 region showed pal to be the most important determinant for genomic RNA packaging and dimerization (30).…”