An Extended Stem-Loop 1 Is Necessary for Human Immunodeficiency Virus Type 2 Replication and Affects Genomic RNA Encapsidation

Abstract

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3 end of the encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3 end of the pal within with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.The 5Ј-untranslated regions of retroviral RNAs contain several structured motifs that are involved in various steps in viral replication, including transcriptional transactivation, splicing, encapsidation, dimerization, and initiation of reverse transcription (for a review, see reference 3). Encapsidation of the genomic RNA in lentiviruses is a very specific process. Two molecules of unspliced RNA are selected for encapsidation over a vast excess of different spliced viral RNA species and cellular RNA. The historical model of retroviral RNA encapsidation has been that viral structural proteins like Gag recognize and bind specific RNA motifs located downstream of the major splice donor site, and thus only present in the unspliced viral RNA, and transfer a pair of these molecules into one newly forming viral particle (for a review, see references 11 and 20).However, most recent studies suggest that the encapsidation signal is not limited to a single RNA element.

SHAPE analysis of the 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization

“…Similar conservation of a DIS comprising a pal sequence has been reported in several retroviruses, including HIV-1, HIV-2, SIV, and FIV (Paillart et al 2002;Russell et al 2004;Lever 2007;Kenyon et al 2008Kenyon et al , 2011. In a number of retroviruses, sequences augmenting RNA dimerization and packaging have been shown to be intermingled (Lanchy et al 2003;Paillart et al 2004;Russell et al 2004;Lanchy and Lodmell 2007;Lever 2007), which is in agreement with the deletion of MPMV pal SL resulting in ablation of RNA packaging (Jaballah et al 2010).…”

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

“…In particular, we showed that the formation of the extended SL1 structure is necessary for HIV-2 replication and genomic RNA encapsidation (Lanchy and Lodmell 2007). Our experiments showed that one important role of the 39 end of pal is to base-pair with the 59-GGAGC motif located downstream of SL1 and that this interaction is necessary for an effective genomic RNA encapsidation to occur (Lanchy and Lodmell 2007). Although a strong selective pressure during viral replication for an adenine FIGURE 7.…”

“…at position 394 was observed upon mutation to a guanine residue (Lanchy and Lodmell 2007), the precise role of the 59 end of pal is less understood. Nevertheless, it is notable that the extended SL1 structure is surrounded by two 59-GGAG motifs, including one in pal (Fig.…”

“…Third, the 39 end of wild-type pal may interact with a 59 GGAGC motif located downstream of SL1 ( Fig. 6B; McCann and Lever 1997;Dirac et al 2001) and form a structure similar to the one found in pal*-444 region species, which we called the extended SL1 (Lanchy and Lodmell 2007).…”

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

“…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). Thus, at this time, it is unclear which RNA element(s) is important for the RNA partner selection process if HIV-2 Gag packages dimeric RNA.…”