HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Chen, Jianbo; Rahman, Sheikh Abdul; Nikolaitchik, Olga A.; Grünwald, David; Sardo, Luca; Burdick, Ryan C.; Plisov, Sergey; Liang, Edward; Tai, Sheldon; Pathak, Vinay K.; Hu, Wei-Shau

doi:10.1073/pnas.1518572113

Cited by 71 publications

(105 citation statements)

References 32 publications

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“…The dynamic interactions between HIV-1 Gag and RNA genome on plasma membrane have been described (29)(30)(31). We have previously studied the population dynamics of HIV-1 RNA on the plasma membrane and found that the presence of Gag protein significantly extends the time that HIV-1 RNA resides near the plasma membrane, indicating that most viral RNAs on the plasma membrane are in Gag-RNA complexes (31).…”

Section: Discussionmentioning

confidence: 99%

“…We have also examined HIV-1 RNA dimerization using live-cell total internal reflection fluorescence microscopy and observed that HIV-1 RNA molecules interact with each other dynamically on the plasma membrane, often when RNAs are associated with Gag signals. Furthermore, a significant number of HIV-1 Gag molecules are required to stabilize the RNA dimer (30).…”

Section: Discussionmentioning

confidence: 99%

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Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Dilley

Nikolaitchik

Galli

et al. 2017

J Virol

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Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly.IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Dilley

Nikolaitchik

Galli

et al. 2017

J Virol

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“…Genomes are initially anchored to the PM by a small number of Gag proteins (~12 copies or fewer) [1–4], where they diffuse laterally and stochastically, then collide to form dimers [5] and become fixed at sites where additional Gag proteins bind [1–6]. Proteins of the cellular protein sorting machinery are also recruited to assembly sites to promote budding [3,7,8].…”

Section: Introductionmentioning

confidence: 99%

Structural and Molecular Determinants of Membrane Binding by the HIV-1 Matrix Protein

Mercredi

Bucca

Loeliger

et al. 2016

Journal of Molecular Biology

115

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Assembly of HIV-1 particles is initiated by the trafficking of viral Gag polyproteins from the cytoplasm to the plasma membrane (PM), where they co-localize and bud to form immature particles. Membrane targeting is mediated by the N-terminally myristoylated matrix (MA) domain of Gag and is dependent on the PM marker phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Recent studies revealed that PI(4,5)P2 molecules containing truncated acyl chains [tr-PI(4,5)P2] are capable of binding MA in an “extended lipid” conformation and promoting myristoyl exposure. Here we report that tr-PI(4,5)P2 molecules also readily bind to non-membrane proteins, including HIV-1 capsid (CA), which prompted us to re-examine MA-PI(4,5)P2 interactions using native lipids and membrane mimetic liposomes and bicelles. Liposome binding trends observed using a recently developed NMR approach paralleled results of flotation assays, although the affinities measured under the equilibrium conditions of NMR experiments were significantly higher. Native PI(4,5)P2 enhanced MA binding to liposomes designed to mimic non-raft-like regions of the membrane, supporting proposals that Gag binding may nucleate raft formation. Studies with bicelles revealed a subset of surface and myr-associated MA residues that are sensitive to native PI(4,5)P2, but cleft residues that interact with the 2′-acyl chains of tr-PI(4,5)P2 molecules in aqueous solution were insensitive to native PI(4,5)P2 in bicelles. Our findings call to question extended-lipid MA:membrane binding models, and instead support a model put forward from coarse-grained simulations indicating that binding is mediated predominantly by dynamic, electrostatic interactions between conserved basic residues of MA and multiple PI(4,5)P2 and phosphatidylserine molecules.

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“…Using imaging techniques, several groups showed that gRNA dimerization precedes the budding of viral particles (18)(19)(20)(21)(22)(23). Indeed, HIV-1 gRNA was found to migrate to the PM as a pre-formed dimer (18,23,24) in association with low-order Gag multimers (25)(26)(27), thus forming a viral ribonucleoprotein (vRNP) (for reviews (6,28,29)). Although the cellular trafficking of the vRNP remains to be precisely described, it was proposed that the viral core was alternatively targeted to late endosomes (30)(31)(32)(33), and the dynein motor function could regulate the vRNP egress on endosomal membranes thus impacting viral production (34).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, it was suggested that stabilization of the RNA dimers occurs at the PM assembly sites (24) thanks to the chaperone activity of Gag (18,19,25,26) .…”

Section: Introductionmentioning

confidence: 99%

The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

Bernacchi

Boutant

Bonzi

et al. 2020

Preprint

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The HIV-1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type (WT) Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs), or a non-myristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm,

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HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Cited by 71 publications

References 32 publications

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Structural and Molecular Determinants of Membrane Binding by the HIV-1 Matrix Protein

The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

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