Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

Jones, Christopher P.; Datta, Siddhartha A.K.; Rein, Alan; Rouzina, Ioulia; Musier‐Forsyth, Karin

doi:10.1128/jvi.01809-10

Cited by 80 publications

(138 citation statements)

References 77 publications

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“…Hairpins were also extended after exposure to a solution of NC prepared as described previously (33). The specific binding sites on TAR RNA appear to be saturated at 50 nM NC (34,35), as discussed further in SI Materials and Methods, section 9.…”

Section: Methodsmentioning

confidence: 98%

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

McCauley

Rouzina

Manthei

et al. 2015

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

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Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA-DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA-DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.single molecule | force spectroscopy | RNA stretching | RNA binding T he transactivation response (TAR) RNA hairpin is a 59-nt sequence in the long-terminal repeat (LTR) of the HIV-1 genome that forms a 24-bp hairpin ( Fig. 1A) (1). This structure is essential in promoting viral transactivator protein (Tat)-mediated transcription. The protein-RNA complex further enhances LTR promoter activity (2). The highly stable TAR hairpin structure that stimulates viral RNA transcription becomes a liability during the early stage of a new infection, as TAR hairpins inhibit the minus-strand transfer step required for reverse transcription (1). To alleviate this inhibition, successful reverse transcription requires a key viral chaperone, the nucleocapsid (NC) protein. In vitro experiments have shown a 3,000-fold stimulation of the rate-limiting step of minus-strand transfer in the presence of NC (3), as NC is required to destabilize TAR RNA and the complementary repeat TAR DNA hairpin to allow subsequent strand annealing (1).HIV-1 NC is only 55 aa long, consisting of two highly conserved CCHC zinc fingers and a basic N terminus (1) (Fig. 1B). The multiple roles of NC during reverse transcription all use the same "chaperone" activity (1), which describes HIV-1 NC's ability to facilitate the rearrangement of nucleic acids into the most stable structures, with the lowest free energy (1). This chaperone activity is characterized by nucleic acid aggregation, duplex destabilization, and rapid kinetics of protein-nucleic acid interactions (3, 4). Aromatic residues in...

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Section: Methodsmentioning

confidence: 98%

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

McCauley

Rouzina

Manthei

et al. 2015

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

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“…5). In non-ψ complexes, MA is partially occupied by nucleic acid binding via the same interface used for PIP 2 binding and, therefore, these complexes have a kinetic barrier to plasma membrane binding that involves MA-RNA dissociation (Shkriabai et al 2006;Chukkapalli et al 2010Chukkapalli et al , 2013Datta et al 2011;Jones et al 2011). We propose that the availability of the MA domain for plasma membrane binding in the Gag molecules bound to ψ-containing gRNA could confer a kinetic advantage for gRNA-Gag complexes to initiate assembly (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…MA interactions with RNA are secondary to its binding to phosphatidylinositol-(4, 5)-bisphosphate (PIP 2 )-containing lipids once it reaches the plasma membrane, whereas NC prefers to bind to RNA (Alfadhli et al 2009;Chukkapalli et al 2010;Jones et al 2011). MA is myristoylated and also contains a basic patch, which enhances its binding to PIP 2 (Chukkapalli et al 2008(Chukkapalli et al , 2010.…”

Section: Thementioning

confidence: 99%

“…1) that are critical for RNA-binding specificity, nucleic acid chaperone activity Darlix et al 2011), and gRNA packaging (Gorelick et al 1999b;Kafaie et al 2008). Investigations into the RNAbinding specificity of Gag have previously studied the binding of the NC protein to RNA and DNA (Dannull et al 1994;Fisher et al 1998Fisher et al , 2006Vuilleumier et al 1999;Avilov et al 2009;Athavale et al 2010), and more recently the binding of Gag to short oligonucleotides has also been investigated (Stephen et al 2007;Wu et al 2010;Jones et al 2011). In this study, we investigate the binding of Gag and NC to longer ∼100-nt RNAs derived from the gRNA 5 ′ UTR.…”

Section: Thementioning

confidence: 99%

“…WT GagΔp6 and variants were prepared as previously described (Datta and Rein 2009;Jones et al 2011). Briefly, the preparation involves ammonium sulfate precipitation and ion-exchange (phosphocellulose resin) purification, followed by size exclusion chromatography.…”

Section: Preparation Of Proteins and Nucleic Acidsmentioning

confidence: 99%

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Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Webb

Jones

Parent

et al. 2013

RNA

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131

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Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z eff ) and nonelectrostatic (i.e., specific) component of binding, K d(1M) . Gag binds to Psi RNA with a dramatically reduced K d(1M) and lower Z eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z eff . These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

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Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: Implications for deltaretroviral assembly

2013

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The matrix (MA) domain of retroviral Gag proteins plays a crucial role in virion assembly. In human immunodeficiency virus type 1 (HIV-1), a lentivirus, the presence of phosphatidylinositol-(4,5)-bisphosphate triggers a conformational change allowing the MA domain to bind the plasma membrane (PM). In this study, the MA protein from bovine leukemia virus (BLV) was used to investigate the mechanism of viral Gag binding to the membrane during replication of a deltaretrovirus. Fluorescence spectroscopy was used to measure the binding affinity of MA for two RNA constructs derived from the BLV genome as well as for single-stranded DNA (ssDNA). The importance of electrostatic interactions and the ability of inositol hexakisphosphate (IP6) to compete with nucleic acids for binding to MA were also investigated. Our data show that IP6 effectively competes with RNA and DNA for BLV MA binding, while [NaCl] of greater than 100 mM is required to produce any observable effect on DNA-MA binding. These results suggest that BLV assembly may be highly dependent on the specific interaction of the MA domain with components of the PM, as observed previously with HIV-1. The mode of MA binding to nucleic acids and the implications for BLV assembly are discussed.

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Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

Cited by 80 publications

References 77 publications

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: Implications for deltaretroviral assembly

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