Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications

Wu, Tsung‐Tsong; Datta, Siddhartha A.K.; Mitra, Mithun; Gorelick, Robert J.; Rein, Alan; Levin, Joseph

doi:10.1016/j.virol.2010.06.042

Cited by 42 publications

(81 citation statements)

References 78 publications

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“…The K 1/2 value reflects a chaperone protein's binding affinity (K d ) and binding site size, as well as cooperativity. For NC (K 1/2 of ϳ7.90), about four times more protein was required to achieve half-maximal annealing relative to the amount of Gag⌬p6 (K 1/2 of ϳ30.2), consistent with previous studies comparing the two proteins (59,75). Gag⌬p6, CANC (K 1/2 of ϳ29.9), and Gag⌬p6-K30,32N (K 1/2 of ϳ26.7) facilitate annealing at lower concen-trations than their monomeric counterparts WM-Gag⌬p6 (K 1/2 of ϳ18.8), WM-CANC (K 1/2 of ϳ12.7), and WM-Gag⌬p6-K30,32N (K 1/2 of ϳ 11.6) ( Table 1).…”

Section: Resultssupporting

confidence: 89%

“…In viruses lacking active protease, tRNA 3 Lys annealing to the PBS still occurs although at reduced levels, suggesting that the precursor protein Gag may act as an NA chaperone and facilitate this process in vivo (34). Indeed, Gag⌬p6, which lacks the C-terminal p6 domain, and other assembly-competent Gag variants can facilitate tRNA annealing and genome dimerization in vitro (21,29,59,75).…”

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confidence: 99%

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

Jones

Datta

Rein

et al. 2011

J Virol

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124

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Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA 3 Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA 3 Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA 3 Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.

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Section: Resultssupporting

confidence: 89%

mentioning

confidence: 99%

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

Jones

Datta

Rein

et al. 2011

J Virol

Self Cite

124

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“…The high affinity of binding may prevent the rapid on-off binding kinetics of NC required for its chaperone activity (Cruceanu et al 2006a;Levin et al 2010). Consistent with this hypothesis, Gag and NCp15 proteins have higher affinities than NC for nucleic acids but their chaperone activity is weaker (Cruceanu et al 2006b;Wu et al 2010;Wang et al 2014).…”

Section: Introductionmentioning

confidence: 73%

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Belfetmi¹,

Zargarian²,

Tisné³

et al. 2016

RNA

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The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.

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“…Since, however, NC-p6 particles had a much higher proportion of irregular cores than wt particles and the irregular cores occupied a larger fraction of the inner virion volume in both variants, a kinetic delay rather than a failure of RNP condensation may be the cause of irregular capsid formation. NC and NC-SP2 have been shown to be significantly more active nucleic acid chaperones than NC-p6 (11,12,27,32,33,36,43,50), and this chaperone activity is needed to condense the RNA genome into the most thermodynamically stable form. The lower chaperone activity of NC-p6 may translate into delayed condensation of the RNP upon maturation and thereby indirectly affect capsid condensation.…”

Section: Discussionmentioning

confidence: 99%

Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity

Marco

Heuser

Glass

et al. 2012

J Virol

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b HIV-1 buds as an immature, noninfectious virion. Proteolysis of its main structural component, Gag, is required for morphological maturation and infectivity and leads to release of four functional domains and the spacer peptides SP1 and SP2. The Nterminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two Cterminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity, and structural maturation. Neither the presence nor the proteolytic processing of SP2 was required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutation of the slow cleavage site between NC and SP2 or deletion of most of SP2 had only a minor effect on infectivity and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6 but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.

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Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications

Cited by 42 publications

References 78 publications

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

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

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity

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