Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Prague Czech Republic

Department of Biotechnology University of Chemistry and Technology Prague Prague Czech Republic

Zobrazit více v PubMed

Briggs JA, Riches JD, Glass B, Bartonova V, Zanetti G, Krausslich HG. 2009. Structure and assembly of immature HIV. Proc Natl Acad Sci U S A 106:11090–11095. doi:10.1073/pnas.0903535106. PubMed DOI PMC

Jouvenet N, Simon SM, Bieniasz PD. 2009. Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles. Proc Natl Acad Sci U S A 106:19114–19119. doi:10.1073/pnas.0907364106. PubMed DOI PMC

Kutluay SB, Bieniasz PD. 2010. Analysis of the initiating events in HIV-1 particle assembly and genome packaging. PLoS Pathog 6:e1001200. doi:10.1371/journal.ppat.1001200. PubMed DOI PMC

Bryant M, Ratner L. 1990. Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc Natl Acad Sci U S A 87:523–527. doi:10.1073/pnas.87.2.523. PubMed DOI PMC

Zhou W, Parent LJ, Wills JW, Resh MD. 1994. Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol 68:2556–2569. doi:10.1128/JVI.68.4.2556-2569.1994. PubMed DOI PMC

Saad JS, Miller J, Tai J, Kim A, Ghanam RH, Summers MF. 2006. Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly. Proc Natl Acad Sci U S A 103:11364–11369. doi:10.1073/pnas.0602818103. PubMed DOI PMC

Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO. 2004. Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proc Natl Acad Sci U S A 101:14889–14894. doi:10.1073/pnas.0405596101. PubMed DOI PMC

Briggs JA, Simon MN, Gross I, Krausslich HG, Fuller SD, Vogt VM, Johnson MC. 2004. The stoichiometry of Gag protein in HIV-1. Nat Struct Mol Biol 11:672–675. doi:10.1038/nsmb785. PubMed DOI

Li S, Hill CP, Sundquist WI, Finch JT. 2000. Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature 407:409–413. doi:10.1038/35030177. PubMed DOI

Pornillos O, Ganser-Pornillos BK, Yeager M. 2011. Atomic-level modelling of the HIV capsid. Nature 469:424–427. doi:10.1038/nature09640. PubMed DOI PMC

Mattei S, Glass B, Hagen WJ, Krausslich HG, Briggs JA. 2016. The structure and flexibility of conical HIV-1 capsids determined within intact virions. Science 354:1434–1437. doi:10.1126/science.aah4972. PubMed DOI

Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI. 1999. Assembly and analysis of conical models for the HIV-1 core. Science 283:80–83. doi:10.1126/science.283.5398.80. PubMed DOI

Borsetti A, Ohagen A, Gottlinger HG. 1998. The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly. J Virol 72:9313–9317. doi:10.1128/JVI.72.11.9313-9317.1998. PubMed DOI PMC

Schur FK, Hagen WJ, Rumlova M, Ruml T, Muller B, Krausslich HG, Briggs JA. 2015. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 A resolution. Nature 517:505–508. doi:10.1038/nature13838. PubMed DOI

Mammano F, Ohagen A, Hoglund S, Gottlinger HG. 1994. Role of the major homology region of human immunodeficiency virus type 1 in virion morphogenesis. J Virol 68:4927–4936. doi:10.1128/JVI.68.8.4927-4936.1994. PubMed DOI PMC

Gross I, Hohenberg H, Wilk T, Wiegers K, Grattinger M, Muller B, Fuller S, Krausslich HG. 2000. A conformational switch controlling HIV-1 morphogenesis. EMBO J 19:103–113. doi:10.1093/emboj/19.1.103. PubMed DOI PMC

Schur FK, Obr M, Hagen WJ, Wan W, Jakobi AJ, Kirkpatrick JM, Sachse C, Krausslich HG, Briggs JA. 2016. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation. Science 353:506–508. doi:10.1126/science.aaf9620. PubMed DOI

Wagner JM, Zadrozny KK, Chrustowicz J, Purdy MD, Yeager M, Ganser-Pornillos BK, Pornillos O. 2016. Crystal structure of an HIV assembly and maturation switch. Elife 5:e17063. doi:10.7554/eLife.17063. PubMed DOI PMC

Wright ER, Schooler JB, Ding HJ, Kieffer C, Fillmore C, Sundquist WI, Jensen GJ. 2007. Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells. EMBO J 26:2218–2226. doi:10.1038/sj.emboj.7601664. PubMed DOI PMC

Zhao G, Perilla JR, Yufenyuy EL, Meng X, Chen B, Ning J, Ahn J, Gronenborn AM, Schulten K, Aiken C, Zhang P. 2013. Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics. Nature 497:643–646. doi:10.1038/nature12162. PubMed DOI PMC

Gres AT, Kirby KA, KewalRamani VN, Tanner JJ, Pornillos O, Sarafianos SG. 2015. STRUCTURAL VIROLOGY. X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability. Science 349:99–103. doi:10.1126/science.aaa5936. PubMed DOI PMC

Ganser-Pornillos BK, Cheng A, Yeager M. 2007. Structure of full-length HIV-1 CA: a model for the mature capsid lattice. Cell 131:70–79. doi:10.1016/j.cell.2007.08.018. PubMed DOI

Byeon IJ, Meng X, Jung J, Zhao G, Yang R, Ahn J, Shi J, Concel J, Aiken C, Zhang P, Gronenborn AM. 2009. Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function. Cell 139:780–790. doi:10.1016/j.cell.2009.10.010. PubMed DOI PMC

Jacques DA, McEwan WA, Hilditch L, Price AJ, Towers GJ, James LC. 2016. HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis. Nature 536:349–353. doi:10.1038/nature19098. PubMed DOI PMC

Bhattacharya A, Alam SL, Fricke T, Zadrozny K, Sedzicki J, Taylor AB, Demeler B, Pornillos O, Ganser-Pornillos BK, Diaz-Griffero F, Ivanov DN, Yeager M. 2014. Structural basis of HIV-1 capsid recognition by PF74 and CPSF6. Proc Natl Acad Sci U S A 111:18625–18630. doi:10.1073/pnas.1419945112. PubMed DOI PMC

Price AJ, Jacques DA, McEwan WA, Fletcher AJ, Essig S, Chin JW, Halambage UD, Aiken C, James LC. 2014. Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly. PLoS Pathog 10:e1004459. doi:10.1371/journal.ppat.1004459. PubMed DOI PMC

Dick RA, Zadrozny KK, Xu C, Schur FKM, Lyddon TD, Ricana CL, Wagner JM, Perilla JR, Ganser-Pornillos BK, Johnson MC, Pornillos O, Vogt VM. 2018. Inositol phosphates are assembly co-factors for HIV-1. Nature 560:509–512. doi:10.1038/s41586-018-0396-4. PubMed DOI PMC

Mallery DL, Marquez CL, McEwan WA, Dickson CF, Jacques DA, Anandapadamanaban M, Bichel K, Towers GJ, Saiardi A, Bocking T, James LC. 2018. IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis. Elife 7:e35335. doi:10.7554/eLife.35335. PubMed DOI PMC

Campbell S, Fisher RJ, Towler EM, Fox S, Issaq HJ, Wolfe T, Phillips LR, Rein A. 2001. Modulation of HIV-like particle assembly in vitro by inositol phosphates. Proc Natl Acad Sci U S A 98:10875–10879. doi:10.1073/pnas.191224698. PubMed DOI PMC

Datta SA, Zhao Z, Clark PK, Tarasov S, Alexandratos JN, Campbell SJ, Kvaratskhelia M, Lebowitz J, Rein A. 2007. Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch. J Mol Biol 365:799–811. doi:10.1016/j.jmb.2006.10.072. PubMed DOI PMC

Mallery DL, Faysal KMR, Kleinpeter A, Wilson MSC, Vaysburd M, Fletcher AJ, Novikova M, Bocking T, Freed EO, Saiardi A, James LC. 2019. Cellular IP6 levels limit HIV production while viruses that cannot efficiently package IP6 are attenuated for infection and replication. Cell Rep 29:3983–3996.e4. doi:10.1016/j.celrep.2019.11.050. PubMed DOI PMC

Hadravova R, Rumlova M, Ruml T. 2015. FAITH—Fast Assembly Inhibitor Test for HIV. Virology 486:78–87. doi:10.1016/j.virol.2015.08.029. PubMed DOI

Dostálková A, Hadravová R, Kaufman F, Křížová I, Škach K, Flegel M, Hrabal R, Ruml T, Rumlová M. 2019. A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors. Sci Rep 9:17076. doi:10.1038/s41598-019-53483-w. PubMed DOI PMC

Ulbrich P, Haubova S, Nermut MV, Hunter E, Rumlova M, Ruml T. 2006. Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins. J Virol 80:7089–7099. doi:10.1128/JVI.02694-05. PubMed DOI PMC

Gross I, Hohenberg H, Huckhagel C, Kräusslich HG. 1998. N-Terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles. J Virol 72:4798–4810. doi:10.1128/JVI.72.6.4798-4810.1998. PubMed DOI PMC

Qualley DF, Lackey CM, Paterson JP. 2013. Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: implications for deltaretroviral assembly. Proteins 81:1377–1385. doi:10.1002/prot.24281. PubMed DOI

Qu K, Glass B, Dolezal M, Schur FKM, Murciano B, Rein A, Rumlova M, Ruml T, Krausslich HG, Briggs JAG. 2018. Structure and architecture of immature and mature murine leukemia virus capsids. Proc Natl Acad Sci U S A 115:E11751–E11760. doi:10.1073/pnas.1811580115. PubMed DOI PMC

von Schwedler UK, Stray KM, Garrus JE, Sundquist WI. 2003. Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol 77:5439–5450. doi:10.1128/jvi.77.9.5439-5450.2003. PubMed DOI PMC

Melamed D, Mark-Danieli M, Kenan-Eichler M, Kraus O, Castiel A, Laham N, Pupko T, Glaser F, Ben-Tal N, Bacharach E. 2004. The conserved carboxy terminus of the capsid domain of human immunodeficiency virus type 1 gag protein is important for virion assembly and release. J Virol 78:9675–9688. doi:10.1128/JVI.78.18.9675-9688.2004. PubMed DOI PMC

Chang YF, Wang SM, Huang KJ, Wang CT. 2007. Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag. J Mol Biol 370:585–597. doi:10.1016/j.jmb.2007.05.020. PubMed DOI

Ganser-Pornillos BK, von Schwedler UK, Stray KM, Aiken C, Sundquist WI. 2004. Assembly properties of the human immunodeficiency virus type 1 CA protein. J Virol 78:2545–2552. doi:10.1128/jvi.78.5.2545-2552.2004. PubMed DOI PMC

Obr M, Hadravová R, DoleŽal M, KříŽová I, Papoušková V, Zídek L, Hrabal R, Ruml T, Rumlová M. 2014. Stabilization of the beta-hairpin in Mason-Pfizer monkey virus capsid protein—a critical step for infectivity. Retrovirology 11:94. doi:10.1186/s12977-014-0094-8. PubMed DOI PMC

Chen K, Piszczek G, Carter C, Tjandra N. 2013. The maturational refolding of the β-hairpin motif of equine infectious anemia virus capsid protein extends its helix α1 at capsid assembly locus. J Biol Chem 288:1511–1520. doi:10.1074/jbc.M112.425140. PubMed DOI PMC

Fuzik T, Ulbrich P, Ruml T. 2014. Efficient Mutagenesis Independent of Ligation (EMILI). J Microbiol Methods 106:67–71. doi:10.1016/j.mimet.2014.08.003. PubMed DOI

Křížová I, Hadravová R, Štokrová J, Günterová J, Doležal M, Ruml T, Rumlová M, Pichová I. 2012. The G-patch domain of Mason-Pfizer monkey virus is a part of reverse transcriptase. J Virol 86:1988–1998. doi:10.1128/JVI.06638-11. PubMed DOI PMC

Campbell S, Vogt VM. 1995. Self-assembly in vitro of purified Ca-Nc proteins from Rous sarcoma virus and human immunodeficiency virus type 1. J Virol 69:6487–6497. doi:10.1128/JVI.69.10.6487-6497.1995. PubMed DOI PMC

Rumlová M, Křížová I, Zelenka J, Weber J, Ruml T. 2018. Does BCA3 play a role in the HIV-1 replication cycle? Viruses 10:212. doi:10.3390/v10040212. PubMed DOI PMC

The Present and Future of Virology in the Czech Republic-A New Phoenix Made of Ashes?

Alteration in DNA-binding affinity of Wilms tumor 1 protein due to WT1 genetic variants associated with steroid - resistant nephrotic syndrome in children

Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses