Article

Identification of a Minimal Region of the HIV-1 5 '-Leader Required for RNA Dimerization, NC Binding, and Packaging

Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 03/2012; 417(3):224-39. DOI: 10.1016/j.jmb.2012.01.033
Source: PubMed

ABSTRACT Assembly of human immunodeficiency virus type 1 (HIV-1) particles is initiated in the cytoplasm by the formation of a ribonucleoprotein complex comprising the dimeric RNA genome and a small number of viral Gag polyproteins. Genomes are recognized by the nucleocapsid (NC) domains of Gag, which interact with packaging elements believed to be located primarily within the 5'-leader (5'-L) of the viral RNA. Recent studies revealed that the native 5'-L exists as an equilibrium of two conformers, one in which dimer-promoting residues and NC binding sites are sequestered and packaging is attenuated, and one in which these sites are exposed and packaging is promoted. To identify the elements within the dimeric 5'-L that are important for packaging, we generated HIV-1 5'-L RNAs containing mutations and deletions designed to eliminate substructures without perturbing the overall structure of the leader and examined effects of the mutations on RNA dimerization, NC binding, and packaging. Our findings identify a 159-residue RNA packaging signal that possesses dimerization and NC binding properties similar to those of the intact 5'-L and contains elements required for efficient RNA packaging.

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    • "To study the role of regions 1, 2 and 3 in the HIV-1 leader RNA, we analyzed a large set of virus mutants (Table 1). These regions are single-stranded in the BMH conformation, but involved in some base pairing interactions in other RNA structure models and isolates (Abbink and Berkhout, 2003; Heng et al., 2012; Lu et al., 2011a, 2011b; Sakuragi et al., 2012; van Bel et al., 2014a; Wilkinson et al., 2008). These mutants were randomly picked from previously created SELEX libraries based on the full-length HIV-1 LAI genome with small segments of randomized sequence (van Bel et al., 2014a). "
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    • "However, within the viral context, deletions have been made to trim down the sequences required for packaging of the HIV-1 genome. A minimal 159-nt RNA sequence that includes SL1–SL3 through the U5:AUG stem, but lacks TAR, Poly(A) and the upper PBS hairpin structure, can dimerize and is competent to bind NC in vitro (Heng et al., 2012). In addition, a 144-nt RNA sequence including a segment of sequence that forms the base of the PBS hairpin (but does not include the primer binding site), SL1, SL3 and SL4 proved to be sufficient to mediate intramolecular dimerization when inserted into an ectopic position in the HIV-1 genome, and was defined as the minimal element required for HIV-1 RNA dimerization in vivo (Sakuragi et al., 2007). "
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