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


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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Available from: Michael F Summers
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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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    ABSTRACT: The untranslated leader of the HIV-1 RNA genome is highly structured and contains multiple replication signals. We probed in detail the sequence requirements of a small single-stranded domain using a combination of in silico, in vitro and in vivo virus experiments. Although 'structure-neutral' mutations can be designed by RNA prediction algorithms, experimental follow-up studies nearly always demonstrate local or regional RNA structure changes. Our results indicate that the wild-type HIV-1 RNA sequence has been selected from total sequence space as a unique solution to present critical replication signals in the context of a complex leader structure with small intervening single-stranded segments. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Sep 2015 · Virology
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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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    ABSTRACT: Viruses often deviate from their hosts in the nucleotide composition of their genomes. The RNA genome of the lentivirus family of retroviruses, including human immunodeficiency virus (HIV), contains e.g. an above average percentage of adenine (A) nucleotides, while being extremely poor in cytosine (C). Such a deviant base composition has implications for the amino acids that are encoded by the open reading frames (ORFs), both in the requirement of specific tRNA species and in the preference for amino acids encoded by e.g. A-rich codons. Nucleotide composition does obviously affect the secondary and tertiary structure of the RNA genome and its biological functions, but it does also influence phylogenetic analysis of viral genome sequences, and possibly the activity of the integrated DNA provirus. Over time, the nucleotide composition of the HIV-1 genome is exceptionally conserved, varying by less than 1% per base position per isolate within either group M, N, or O during 1983–2009. This extreme stability of the nucleotide composition may possibly be achieved by negative selection, perhaps conserving semi-stable RNA secondary structure as reverse transcription would be significantly affected for a less A-rich genome where secondary structures are expected to be more stable and thus more difficult to unfold. This review will discuss all aspects of the lentiviral genome composition, both of the RNA and of its derived double-stranded DNA genome, with a focus on HIV-1, the nucleotide composition over time, the effects of artificially humanized codons as well as contributions of immune system pressure on HIV nucleotide bias.
    Full-text · Article · Nov 2012 · Retrovirology
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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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