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

The 5′-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. The human immunodeficiency virus type 1 (HIV-1) encapsidation signal is multipartite, with structural elements located across the 5′-untranslated and Gag-encoding regions (4, 6, 31). Similarly, HIV-2 and simian immunodeficiency virus (SIV) RNA encapsidation motifs have been localized either downstream of the major splice donor site (38), upstream (16, 32, 39, 46), or both (1, 37). A hallmark of retroviral RNA encapsidation is that two molecules of genomic RNA are encapsidated per viral particle (for a review, see references 14, 34, and 41). Gentle extraction and deproteinization of virions revealed that the two encapsidated RNA molecules form a dimer through noncovalent RNA-RNA interactions (28). Electron microscopy studies with increasing denaturation identified the 5′ ends of the genomic RNA molecules as the location of the strongest RNA-RNA interaction in dimers isolated from viral particles; this interaction was named the dimer linkage structure (see reference 2 for the original study and reference 19 for a study with HIV-1). In HIV-1, a strong candidate involved in the formation of the dimer linkage structure was identified in vitro upstream of the major splice donor site. This element was named the dimerization initiation site or stem-loop 1 (SL1) (18, 25, 33, 35, 45). Besides its role in genomic RNA dimerization, the SL1 sequence was shown to be involved in other stages of HIV-1 replication, notably encapsidation (see Table ​Table11 in reference 34). TABLE 1. Changes in the ψ-SL1 region occurring during long-term culture of strong and weak pal-defective viruses We previously presented evidence that the ψ encapsidation signal interferes with SL1-mediated HIV-2 RNA dimerization in vitro (23). The sequence responsible is a 10-nucleotide (nt) palindromic sequence (pal) located at the 3′ end of ψ. The pal motif was also shown to mediate RNA dimerization upon binding of an antisense oligonucleotide directed against SL1 (23). We and others showed that the dimerization of HIV-2 leader RNA is regulated in vitro by a long-distance intramolecular interaction between nucleotides located at the 5′ end of U5 and nucleotides encompassing the gag translation initiation site (10, 24). Thus, several short- and long-distance interactions influence the presentation of the ψ-SL1 region in HIV-2 leader RNA in vitro. Since the pal motif is located within the ψ encapsidation signal, is phylogenetically conserved (see Discussion), and can influence dimerization of HIV-2 leader RNA fragments in vitro, we have analyzed in this study the function of HIV-2 pal during replication in permissive C8166 cells. We also used long-term culture of defective viruses and their phenotypic reversion to gain insight into the necessity of the palindromic sequence and functionally important secondary structure interactions. In particular, we show that the defects in viral replication for some pal-mutated viruses can be compensated for by nucleotide changes in or close to the mutated pal element. Our results provide evidence for the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3′ end of the 10-nt pal element in ψ with a motif located downstream of SL1. Our observations also suggest that formation of the extended SL1 during viral replication positively affects HIV-2 genomic RNA encapsidation, although a palindromic sequence is not strictly required for viral viability.