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

Abstract

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. Although 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 MS to investigate RNA binding of wild-type (WT) 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. The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. Although 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 MS to investigate RNA binding of wild-type (WT) 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. The ability to specifically select the viral genomic RNA (gRNA) for packaging into the assembling virus particle is absolutely necessary for specific replication of HIV-1 and other retroviruses. This selection is critical as the gRNA is surrounded by a great excess of cellular RNAs, and these RNAs can also be packaged under certain conditions. Approximately 2500 copies of the viral structural protein (“Gag”) assemble around a gRNA dimer forming an immature virion (1Bieniasz P. Telesnitsky A. Multiple, switchable protein:RNA interactions regulate human immunodeficiency virus type 1 assembly.Annu. Rev. Virol. 2018; 5 (30048218): 165-18310.1146/annurev-virology-092917-043448Crossref PubMed Scopus (37) Google Scholar, 2Sundquist W.I. Kräusslich H.-G. HIV-1 assembly, budding, and maturation.Cold Spring Harb. Perspect. Med. 2012; 2 (22762019): a00692410.1101/cshperspect.a006924Crossref PubMed Scopus (501) Google Scholar, 3Turner B.G. Summers M.F. Structural biology of HIV.J. Mol. Biol. 1999; 285 (9878383): 1-3210.1006/jmbi.1998.2354Crossref PubMed Scopus (328) Google Scholar, 4Wilk T. Fuller S.D. Towards the structure of the human immunodeficiency virus: Divide and conquer.Curr. 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The 5′ regulates many stages of the viral life cycle, including genome and of reverse transcription, as well as In to the 5′ is composed of structural the the and the binding Psi is composed of with a within (6Kuzembayeva M. Dilley K. Sardo L. Hu W.-S. Life of Psi: How full-length HIV-1 RNAs become packaged genomes in the viral particles.Virology. 2014; 454–455 (24530126): 362-37010.1016/j.virol.2014.01.019Crossref PubMed Scopus (92) Google Scholar, A. RNA Packaging in HIV.Trends Microbiol. 2019; 27 (31085095): 715-72310.1016/j.tim.2019.04.003Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, S.C. K. S. V. S. A. A. J. S. D.A. M. of the HIV-1 RNA packaging 2015; PubMed Scopus Google Scholar). studies that wild-type (WT) dimeric Psi RNA and a Psi by Gag with specificity in vitro (9Comas-Garcia M. Datta S.A. Baker L. Varma R. Gudla P.R. Rein A. 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H. and for of in 2014; PubMed Scopus (160) Google Scholar, RNA structure by of at RNA and of by A. A. E. of RNA Scopus Google Scholar). Here, we used a Psi (Fig. that to high Gag binding and the or as the non-Psi sequence (Fig. as this RNA to be by Gag binding specificity J.A. Jones C.P. Parent L.J. Rouzina I. Musier-Forsyth K. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging.RNA. 2013; 19 (23798665): 1078-108810.1261/rna.038869.113Crossref PubMed Scopus (67) Google Scholar). The of in vitro binding and RNA packaging support the that Gag–Gag interactions are by specific binding to Psi RNA and that this is of the p6 domain. MS used to the of the Gag in this (Fig. 1A). The for the and dimeric forms of Gag the the high of the and the binding of and We the of Gag proteins at in the of The presence of the p6 domain a on the a of dimer observed in the of GagΔp6 at to Gag at 9 and This is with a that Gag has a to to GagΔp6 H.S. Khoo K.K. Garvey M. Waddington L. Leis A. Hijnen M. Velkov T. Dumsday G.J. McKinstry W.J. Mak J. The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV.PLoS Pathog. 2017; 13 (28222188): e100622110.1371/journal.ppat.1006221Crossref PubMed Scopus (26) Google Scholar). a of observed for both proteins (Fig. and at both proteins (Fig. and The presence of the p6 domain the and The observed for Gag is to the presence of the p6 domain J. W. A. A. E. M. F. R. P. in protein 2017; 8 PubMed Scopus Google Scholar). on the dimer interface in the C-terminal domain of as (Fig. and and S. H. W.I. C.P. of the domain of the HIV-1 capsid PubMed Scopus Google Scholar, S. S.J. M. J. Rein A. Interactions between HIV-1 Gag in Mol. Biol. PubMed Scopus Google Scholar). We also the of Gag in the presence of Psi RNA and a control RNA, (Fig. Although is a the used of RNA and of Gag. of Gag not in binding under the ionic used these protein VLP by using a in Gag–RNA interactions are interactions are more than interactions, because of the presence of binding (9Comas-Garcia M. Datta S.A. Baker L. Varma R. Gudla P.R. Rein A. Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.eLife. 2017; 6 (28726630): e2705510.7554/eLife.27055Crossref PubMed Scopus (39) Google Scholar, 10Webb J.A. Jones C.P. Parent L.J. Rouzina I. Musier-Forsyth K. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging.RNA. 2013; 19 (23798665): 1078-108810.1261/rna.038869.113Crossref PubMed Scopus (67) Google Scholar). Thus, the used in the viral assembly nucleation complexes to be observed VLP assembly. is at excess RNA in is the for all (Fig. and and Both Gag and GagΔp6 complexes with These complexes found to contain of Gag and a of (Fig. and and and In in the presence of Psi these proteins complexes of copies of Gag to of Psi RNA, with of complexes (Fig. and and and The of complexes with be by Gag binding to RNA as a dimer by Gag binding to The of Gag is mediated by an interface within complexes of Gag and RNA via Gag–Gag as well as Gag–RNA interactions, we a Gag lacking this complexes of and of RNA for both and Psi RNAs (Fig. and and the that Psi RNA binding promotes of Gag than Gag binding to binding in Psi RNA. these that the primarily Gag protein by using the in the presence of RNA. The of Gag dimer in the presence of Psi RNA from changes in the Gag upon NC domain binding to we not changes in studies have that the NC domain can different upon binding to different RNAs Wu L. Summers M.F. of the HIV-1 nucleocapsid protein to the recognition PubMed Scopus Google Scholar, Wu Summers M.F. structure of the HIV-1 nucleocapsid protein to of the packaging Implications for genome Mol. Biol. PubMed Scopus Google Scholar). Thus, it is that the of the RNA the NC when regulate the of a Gag investigate in binding specificity between Gag and we binding T. Rouzina I. Musier-Forsyth K. Fluorescence to Mol. Biol. 2015; PubMed Scopus Google Scholar). interaction between protein and RNA, mediated primarily by interactions, at a than that also specific interactions (Fig. and and for can be from this and The at 1 the of to is a of the of from RNA upon protein binding P.D. on Mol. Biol. 107 PubMed Scopus Google Scholar, I. V.A. binding of to using the PubMed Scopus Google Scholar). this we that the binding of GagΔp6 to Psi is more than its binding to J.A. Jones C.P. Parent L.J. Rouzina I. Musier-Forsyth K. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging.RNA. 2013; 19 (23798665): 1078-108810.1261/rna.038869.113Crossref PubMed Scopus (67) Google Scholar). In with these the for GagΔp6 binding to Psi and RNAs are and and the of are and In the of the for Psi and are and and the are and Thus, we that the presence of the p6 domain does not the specificity of Gag Psi RNA (Fig. using in of the also that binding between Psi and RNA are observed under and We also the ability of Gag with and the p6 domain to selectively gRNA in human cells. cells with Gag and a an This Psi and be selectively packaged by Gag. Packaging of the RNA in virus particles by using a Gag in the Gag region lacked the p6 domain. The of p6 upon virus particle by of viral from the of the GagΔp6 produced a of than Gag. The of with GagΔp6 of that in of Gag. of Gag and GagΔp6 in of the cells (Fig. that the of the proteins of cells GagΔp6 (Fig. many and as well as particles at the of these are with the of p6 in virus particle release E.O. HIV-1 assembly, release and maturation.Nat. Rev. Microbiol. 2015; 13 (26119571): 484-49610.1038/nrmicro3490Crossref PubMed Scopus Google Scholar, E.O. HIV-1 gag in the virus life PubMed Scopus Google Scholar, J.M. E.O. The domain of human immunodeficiency virus type 1 p6 promotes virus release in a Virol. PubMed Scopus Google Scholar). RNA also from the and for the the of these for the in virus particle are in is that the of RNA to Gag protein in the viral from GagΔp6 is of the in the Gag The of RNA in the GagΔp6 that GagΔp6 has the ability to selectively Psi-containing this as with cellular mRNA in the of packaging of gRNA S.J. J. T. S. Rein A. and packaging of cellular RNAs in Virol. PubMed Scopus Google Scholar). the ability to selectively Psi-containing RNA be but in between these we the packaging of a of the lacking Psi in the of Psi the packaging of the RNA by GagΔp6 well as by that the in the of that with the These data that the GagΔp6 still to a very the ability to Psi-containing RNA. The p6 domain in the interactions of Gag with the cellular during virus particle from cells. Thus, it is that the of this interaction in GagΔp6 is responsible for the in packaging of RNA. as or virions can be in of cells (Fig. of these are packaging gRNA, or the GagΔp6 particles to RNA from this we also packaging of the RNA by full-length Gag in the the in p6 that with the with J.M. E.O. The domain of human immunodeficiency virus type 1 p6 promotes virus release in a Virol. PubMed Scopus Google Scholar). This the of p6, the of particle (Fig. and in the of particles (Fig. in these also a of RNA to Gag protein than by Gag. with and we also found that of Psi from the its packaging by the that the packaging of the intact by this Gag is (Fig. the data that the in RNA packaging by GagΔp6 is a of its interaction with and that it has not specificity in its interactions with RNA. has been that virions from GagΔp6 or are in reverse and J.M. E.O. The domain of human immunodeficiency virus type 1 p6 promotes virus release in a Virol. PubMed Scopus Google it that gRNA, these viral is from or within the virions during the between assembly and release from the The data to the interactions between the structural protein of HIV-1 virus particles, and RNA. the presence or of the p6 domain has on Psi interactions in in vitro binding and does not to the packaging of viral RNA in for an to the with this Gag Gag or is when it binds the Psi RNA packaging but dimer is observed when it binds a control RNA (Fig.

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