Efficient support of virus-like particle assembly by the HIV-1 packaging signal

Comas-García, Mauricio; Kroupa, Tomáš; Datta, Siddhartha A.K.; Harvin, Demetria; Hu, Wei-Shau; Rein, Alan

doi:10.7554/elife.38438

Cited by 34 publications

(43 citation statements)

References 24 publications

(45 reference statements)

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“…Thus, the selection of Psi + RNA during virus assembly is evidently not due to a uniquely high affinity of Gag for this RNA. We and others have suggested that binding to Psi induces a nucleating event, such as formation of a small Gag oligomer, more efficiently than binding to other RNAs, and that this difference might underlie the selective packaging of Psi + RNA (7,11,12,(51)(52)(53). The present results provide very strong support for this fundamental concept.…”

Section: Discussionsupporting

confidence: 77%

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

Sarni

Biswas

Liu

et al. 2020

Journal of Biological Chemistry

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The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

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Section: Discussionsupporting

confidence: 77%

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

Sarni

Biswas

Liu

et al. 2020

Journal of Biological Chemistry

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show abstract

“…Thus, the selection of Psi + RNA during virus assembly is evidently not due to a uniquely high affinity of Gag for this RNA. We and others have suggested that binding to Psi induces a nucleating event, such as formation of a small Gag oligomer, more efficiently than binding to other RNAs, and that this difference might underlie the selective packaging of Psi + RNA (11,12,(50)(51)(52). The present results provide very strong support for this fundamental concept.…”

Section: Discussionsupporting

confidence: 78%

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

Sarni

Biswas

Liu

et al. 2020

Preprint

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The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically nucleates Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

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“…There were also no detected differences from WT protein in the binding of 8N Gag and WM Gag to dimeric Ψ RNA at 300 mM NaCl. We believe that the difference could be caused by the use of shorter RNA constructs and a different measuring buffer; a direct comparison of the fluorescence quenching and microscale thermophoresis binding experiments with a 400 nt long Ψ RNA construct by Comas-Garcia et al [15] found negligible differences between the two experimental techniques. It is also possible that thermophoresis is inherently more sensitive, with regard to weak binding, than the quenching assay.…”

Section: Discussionmentioning

confidence: 98%

“…A previous SHAPE structure-probing analysis of the HIV-1 5 UTR revealed that the main NC interaction domain within Ψ consists of seven short stretches containing unpaired guanosines that are recognized by the NC zinc fingers [13]. It has been shown that mutating these unpaired guanosines interfered with the packaging of the RNA in virus-producing cells [14] and led to the less efficient assembly of the viral particles in vitro [15].…”

Section: Introductionmentioning

confidence: 99%

“…Especially, how is it possible that the viral RNA is packaged into the newly formed virus almost exclusively? We have reported that Gag protein assembles much more efficiently upon interaction with the viral genomic RNA [15] than with control RNAs, and suggested that this is the explanation for selective packaging of genomic RNA in vivo, where it is surrounded by a great excess of other RNAs [1]. The mechanism of this selective packaging is still not well understood.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA

Kroupa

Datta

Rein

2020

Viruses

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Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.

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Efficient support of virus-like particle assembly by the HIV-1 packaging signal

Cited by 34 publications

References 24 publications

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA

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