Structure of the HIV-1 RNA packaging signal

Keane, Sarah C.; Heng, Xiao; Lu, Kun; Kharytonchyk, Siarhei; Ramakrishnan, Venkateswaran; Carter, Gregory; Barton, Shawn; Hosic, Azra; Florwick, Alyssa; Santos, Justin; Bolden, Nicholas C; McCowin, Sayo; Case, David A.; Johnson, B. V.; Salemi, Marco; Telesnitsky, Alice; Summers, Michael F.

doi:10.1126/science.aaa9266

Cited by 234 publications

(328 citation statements)

References 51 publications

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“…1; Jaballah et al 2010;Aktar et al 2013). LRIs have been found to be conserved in several retroviruses, including HIV-1 and 2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and mouse mammary tumor virus (MMTV) (Paillart et al 2002(Paillart et al , 2004bAbbink and Berkhout 2003;Kenyon et al 2008Kenyon et al , 2011Aktar et al 2014;Siegfried et al 2014;Keane et al 2015;Smyth et al 2015;Tran et al 2015). Despite the fact that there are substantial sequence heterogeneities among human, simian, and feline lentiviruses, the preservation of such LRIs in these lentiviruses suggest they play important function(s) in the retroviral life cycle (Paillart et al 2002;Kenyon et al 2008Kenyon et al , 2011.…”

Section: Discussionmentioning

confidence: 99%

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

Kalloush¹,

Vivet-Boudou²,

Ali³

et al. 2016

RNA

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MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2 ′ hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5 ′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. Keywords: retroviruses; Mason-Pfizer monkey virus (MPMV); RNA secondary structure; RNA packaging and dimerization; long-range interactions (LRI); SHAPE (selective 2 ′ hydroxyl acylation analyzed by primer extension)

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Section: Discussionmentioning

confidence: 99%

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

Kalloush¹,

Vivet-Boudou²,

Ali³

et al. 2016

RNA

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“…Similarly, mutations that prevent dimerization of the recombinant 5′-L RNA of the Moloney murine leukemia virus (MLV) block packaging (40), suggesting that dimerization-dependent control mechanisms are conserved among evolutionarily distant retroviruses (40,41). MLV-derived vectors harboring monomer-stabilizing mutations remain unpackaged in the absence of competing wild-type leader RNAs (40), whereas mutations that shift the equilibrium of the HIV-1 5′-L in favor of the monomer but do not prevent dimerization do not prevent the HIV-1 RNA from functioning as both genome and mRNA in the absence of wild-type competition (26,39,42). These and other observations support a mechanism in which a single viral transcript equilibrates between structures associated with alternate replication functions (9,12,16,26,29).…”

Section: Discussionmentioning

confidence: 99%

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

Kharytonchyk

Monti

Smaldino

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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119

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The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3-R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5′ capped guanosine ( Cap 1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines ( Cap 2G or Cap 3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer-dimer equilibrium properties of Cap 1G, Cap 2G, and Cap 3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap 1G 5′-leader RNA adopts a dimeric structure, the Cap 2G and Cap 3G 5′-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimerpromoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5′ guanosine.HIV-1 | 5′-leader | transcription | RNA | structure

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“…We therefore do not discuss the use of NMR to study nucleic acid dynamics (Kimsey et al 2015) and structure (Keane et al 2015), or quantitative analysis of protein dynamics using solid state NMR spectroscopy (Lewandowski, 2013;Ullrich & Glaubitz, 2013). We also do not cover the use of NMR to study protein folding or intrinsically disordered proteins Wright & Dyson, 2015), although it is important to note that both fields are relevant to protein function.…”

Section: Introductionmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

148

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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Structure of the HIV-1 RNA packaging signal

Cited by 234 publications

References 51 publications

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

Protein dynamics and function from solution state NMR spectroscopy

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