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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    • "Inside the HIV-1 virus particle, two copies of its single-stranded RNA genome are packaged, together with virus-encoded proteins and cellular RNA molecules including tRNAlys3 that acts as a primer for reverse transcription [21,22]. Packaging signals in the viral RNA are essential for its incorporation into an assembling virion particle [23], as are signals for RNA dimerization and nucleocapsid (NC) protein binding [24]. The NC protein covers the complete viral RNA genome and could thus be a sensor of nucleotide composition (for a review, see [25]). "
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    • "In agreement with this, we observed efficient packaging of HIV-rtTA genomic RNAs in which TAR was truncated (this study), completely deleted or replaced by a non-related hairpin (ER2 and ER3 variants described in [30]; results not shown). Moreover, Heng et al. very recently demonstrated that TAR deletion did not significantly affect RNA dimerization, NC binding and RNA packaging [58]. However, as we show in this study, opening of the 5′ TAR hairpin perturbs the leader RNA structure and affects both dimerization and packaging of the viral RNAs. "
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    ABSTRACT: The TAR hairpin is present at both the 5' and 3' end of the HIV-1 RNA genome. The 5' element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5' stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. We demonstrate that opening the 5' TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.
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