Building Complexity: Making and Breaking Synthetic Subunits of the HIV Capsid
2019; Cell Press; Volume: 26; Issue: 2 Linguagem: Inglês
10.1016/j.chom.2019.07.011
ISSN1934-6069
Autores Tópico(s)Protein Structure and Dynamics
ResumoIn this issue of Cell Host & Microbe, Summers et al., 2019Summers B.J. Digianantonio K.M. Smaga S.S. Huang P.-T. Zhou K. Gerber E.E. Wang W. Xiong Y. Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms.Cell Host Microbe. 2019; 26 (this issue): 203-216Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar use protein engineering to generate a toolbox of HIV-1 capsid oligomers. In an accompanying Cell Reports paper, Huang et al., 2019Huang P.-T. Summers B.J. Xu C. Perilla J.R. Malikov V. Naghavi M.H. Xiong Y. FEZ1 is recruited to a conserved cofactor site on capsid to promote HIV-1 trafficking.Cell Reports. 2019; 28https://doi.org/10.1016/j.celrep.2019.07.079Scopus (36) Google Scholar use these oligomers to determine how the capsid engages the kinesin-1 adaptor protein FEZ1. In this issue of Cell Host & Microbe, Summers et al., 2019Summers B.J. Digianantonio K.M. Smaga S.S. Huang P.-T. Zhou K. Gerber E.E. Wang W. Xiong Y. Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms.Cell Host Microbe. 2019; 26 (this issue): 203-216Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar use protein engineering to generate a toolbox of HIV-1 capsid oligomers. In an accompanying Cell Reports paper, Huang et al., 2019Huang P.-T. Summers B.J. Xu C. Perilla J.R. Malikov V. Naghavi M.H. Xiong Y. FEZ1 is recruited to a conserved cofactor site on capsid to promote HIV-1 trafficking.Cell Reports. 2019; 28https://doi.org/10.1016/j.celrep.2019.07.079Scopus (36) Google Scholar use these oligomers to determine how the capsid engages the kinesin-1 adaptor protein FEZ1. The HIV-1 capsid undergoes a number of transformative changes during the viral replicative cycle. The infectious form, a fullerene cone produced following a process of metamorphosis inside budded virions, is perhaps one of the most iconic of viral structures (Ganser et al., 1999Ganser B.K. Li S. Klishko V.Y. Finch J.T. Sundquist W.I. Assembly and analysis of conical models for the HIV-1 core.Science. 1999; 283: 80-83Crossref PubMed Scopus (506) Google Scholar). It is assembled from a single viral protein. Exactly how this assembly is accomplished is still unclear, although recent work suggests that assembly may be catalyzed by the abundant polyanion inositol hexakisphosphate (IP6), which is selectively incorporated into virions during budding (Dick et al., 2018Dick R.A. Zadrozny K.K. Xu C. Schur F.K.M. Lyddon T.D. Ricana C.L. Wagner J.M. Perilla J.R. Ganser-Pornillos B.K. Johnson M.C. et al.Inositol phosphates are assembly co-factors for HIV-1.Nature. 2018; 560: 509-512Crossref PubMed Scopus (111) Google Scholar, Mallery et al., 2018Mallery D.L. Márquez C.L. McEwan W.A. Dickson C.F. Jacques D.A. Anandapadamanaban M. Bichel K. Towers G.J. Saiardi A. Böcking T. James L.C. IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis.Elife. 2018; 7https://doi.org/10.7554/eLife.35335Crossref Scopus (89) Google Scholar). The capsomers are arranged into a fullerene cone consisting largely of hexamers with 5 pentamers at one end and 7 at the other. Other structures can be formed both in vitro and in virio, including partial cones, spheres, and tubes; but control of this morphology and its importance is still largely unclear. To study such a complex structure, Summers et al., 2019Summers B.J. Digianantonio K.M. Smaga S.S. Huang P.-T. Zhou K. Gerber E.E. Wang W. Xiong Y. Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms.Cell Host Microbe. 2019; 26 (this issue): 203-216Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, this issue of Cell Host & Microbe) used its component monomer like a LEGO brick to build ever-larger assemblies (Figure 1A). This was made possible by a plethora of clever engineering tricks to stabilize oligomers that normally don’t exist in isolation, including disulphides to staple subunits together, a trimerization domain to induce oliogomerization, and a covalent reactive tag system to capture weak interfaces. The result is an exploded-view breakdown of the HIV-1 capsid into components that, while not strictly intermediates in the assembly pathway, showcase the individual interfaces that hold the structure together. The power of these synthetic assemblies to study capsid:host interaction is demonstrated in an accompanying paper from the same lab in Cell Reports (Huang et al., 2019Huang P.-T. Summers B.J. Xu C. Perilla J.R. Malikov V. Naghavi M.H. Xiong Y. FEZ1 is recruited to a conserved cofactor site on capsid to promote HIV-1 trafficking.Cell Reports. 2019; 28https://doi.org/10.1016/j.celrep.2019.07.079Scopus (36) Google Scholar). In this study, the authors investigate the kinesin-1 adaptor protein FEZ1 and show that capsid hexamers are the minimal assembly capable of supporting FEZ1 binding. Dissection of the interaction through mutagenesis reveals that it is driven primarily by a stretch of 5 glutamates. These glutamates interact with a recently identified binding site located inside capsid hexamers, comprising a ring of six arginines (R18) that recruits the assembly factor IP6 and the deoxyribonucleoside triphosphates required to drive viral DNA synthesis (Jacques et al., 2016Jacques D.A. McEwan W.A. Hilditch L. Price A.J. Towers G.J. James L.C. HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis.Nature. 2016; 536: 349-353Crossref PubMed Scopus (130) Google Scholar, Mallery et al., 2018Mallery D.L. Márquez C.L. McEwan W.A. Dickson C.F. Jacques D.A. Anandapadamanaban M. Bichel K. Towers G.J. Saiardi A. Böcking T. James L.C. IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis.Elife. 2018; 7https://doi.org/10.7554/eLife.35335Crossref Scopus (89) Google Scholar). The earliest view of the HIV capsid was that it serves to package the viral genome and essential enzymes like reverse transcriptase and integrase. Other than interaction with the isomerase cylclophilin A, which binds to exposed loops on the external surface of the capsid (Luban et al., 1993Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B.Cell. 1993; 73: 1067-1078Abstract Full Text PDF PubMed Scopus (703) Google Scholar), it was not believed to interact with host molecules. This view was challenged by structural and biophysical approaches that identified conserved hot-spots and interfaces on the capsid used to bind and recruit essential cofactors (Price et al., 2014Price A.J. Jacques D.A. McEwan W.A. Fletcher A.J. Essig S. Chin J.W. Halambage U.D. Aiken C. James L.C. Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.PLoS Pathog. 2014; 10: e1004459Crossref PubMed Scopus (172) Google Scholar). One of the difficulties in studying cofactor interactions with the capsid has been recreating multimeric assemblies with recombinant protein and capturing native interfaces and symmetry axes (Figure 1B)—such as those within and between hexamers and pentamers. Many of the recent discoveries of new capsid interfaces have only been possible due to pioneering engineering work by Owen and Barbie Pornillos, in which hexamers and pentamers were stabilized by intersubunit disulphide bridges (Pornillos et al., 2010Pornillos O. Ganser-Pornillos B.K. Banumathi S. Hua Y. Yeager M. Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus.J. Mol. Biol. 2010; 401: 985-995Crossref PubMed Scopus (87) Google Scholar). However, as these assemblies are themselves subunits in the larger capsid lattice, the study of host proteins that recognize interfaces between hexamers (or between hexamers and pentamers) or those that bind to capsomers in neighboring hexamers has remained problematic. An example of this is the restriction factor TRIM5α and its ortholog TRIMCyp. Despite having weak affinity for capsid monomers, TRIM5α covers the capsid with an inhibitory lattice by binding across hexamers (Ganser-Pornillos et al., 2011Ganser-Pornillos B.K. Chandrasekaran V. Pornillos O. Sodroski J.G. Sundquist W.I. Yeager M. Hexagonal assembly of a restricting TRIM5alpha protein.Proc. Natl. Acad. Sci. USA. 2011; 108: 534-539Crossref PubMed Scopus (166) Google Scholar). In their study, Summers et al., 2019Summers B.J. Digianantonio K.M. Smaga S.S. Huang P.-T. Zhou K. Gerber E.E. Wang W. Xiong Y. Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms.Cell Host Microbe. 2019; 26 (this issue): 203-216Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar took the requirement for multi-hexamer interaction of TRIM5α, and the related restriction factor MxB, as the impetus to design capsid assemblies that capture these missing inter-hexamer binding sites. The authors began by mixing monomers that contained only some of the established disulphide bridges to make cross-linked oligomers representing 1/3 or 1/2 hexamers and validated their structures by X-ray crystallography (Figure 1A). Importantly, specific subunits within these partial hexamers could be mutated to dissect binding modes of host proteins interacting with multiple sites within a single hexamer. Next, the authors fused their 1/3 hexamers to a trimerization domain to achieve a “trimer of dimers”. A crystal structure confirmed that this oligomer achieved a native-like 3-fold inter-hexamer interface. To study binders that require larger pieces of capsid lattice, the authors used a clever piece of protein engineering to tackle the challenge of connecting multiple hexamers in a variety of arrangements. They exploited a covalent reactive tag system comprising two components called “SpyCatcher” and “SpyTag”. One hexamer is assembled with a subunit containing the “catcher” and another has a subunit with the “tag”. After mixing, the two hexamers are locked together when catcher and tag react. The approach is equivalent to taking pre-existing assemblies of LEGO pieces and joining them together using a K’Nex-like system (Figure 1A). The authors show how mixing different ratios of catcher and tag hexamers can be used to create both di- and tri- hexamer units. A complete assembly recapitulating a central hexamer fully bound by 6 other hexamers proved the most challenging. Even use of a nanobody to stabilize this hepta-hexamer was only partially effective, and further engineering will be required before it can be usefully employed. However, with the assemblies that were formed, the authors were able to show that the restriction factor MxB binds across the 3-fold hexamer axis, while TRIMCyp binds capsid monomers both within and across neighboring hexamers. These results nicely support existing data that these antiviral proteins engage with the capsid lattice rather than individual hexamers to inhibit infection. In their second paper, the same group investigates the kinesin-1 adaptor protein FEZ1, which binds capsid and has been implicated in transporting particles to the nucleus. Xiong’s team uses their partial capsid assemblies to show that a hexamer contains the binding site. FEZ1 is a highly negatively charged protein with polyglutamate repeats, suggesting it could bind the positively charged arginine ring that is only present in hexamers. Sure enough, mutation of the five sequential glutamates in FEZ1 or of the six arginines in the hexamer reduced binding in gel filtration assays. Furthermore, binding of FEZ1 was significantly reduced in the presence of physiological concentrations of the polyanions ATP and IP6, which also bind to the ring of six arginines in the hexamer. FEZ1 contains additional negatively charged segments and these are also shown to possess weak binding activity under low salt conditions, suggesting that FEZ1 may benefit from avidity and interact with multiple R18 rings simultaneously (Figure 1C). FEZ1 has previously been shown to promote the movement of HIV-1 virions along microtubules (Malikov et al., 2015Malikov V. da Silva E.S. Jovasevic V. Bennett G. de Souza Aranha Vieira D.A. Schulte B. Diaz-Griffero F. Walsh D. Naghavi M.H. HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus.Nat. Commun. 2015; 6: 6660Crossref PubMed Scopus (82) Google Scholar). Surprisingly, expression of FEZ1 with mutation of the polyglutamate motif required for high affinity hexamer binding did not alter virion transport and only modestly decreased HIV infection in cells depleted of endogenous FEZ1. In contrast, overexpression of a FEZ1 construct with glutamates in the central region mutated failed to support infection. Why mutation of the penta-glutamate motif essential for binding in vitro does not prevent infectivity or transport while mutation of additional weakly interacting glutamates does remains an open question. There has been a rapid increase in the number of reported HIV-1 capsid binding proteins and more binders are likely to be discovered in the next few years. The generation of a toolbox of capsid constructs will greatly facilitate dissection of their binding mode and aid structural characterization. Future work will be needed to build on this toolbox to understand the interfaces within and between pentamers (and neighboring hexamers) and explore potential novel binding sites and pentamer-specific cofactors. Meanwhile, the binding of FEZ1 and of a minimal fragment of just 11 amino acids containing the polyglutamate motif illustrates the strength of interaction that can be achieved with the HIV-1 capsid R18 ring via a stretch of just five polyglutamates. We note that polyglutamate-containing proteins are highly enriched in the human proteome. Our analysis of proteins currently in Ensemble sugests that more than 1,300 proteins contain at least one stretch of five polyglutamates. This is in contrast to stretches of five histidines or threonines, which are found in only 106 and 154 proteins, respectively. Hydrophobic patches and “sticky” proteins, such as HIV-1 integrase, are well known to confound the investigation of specific protein-protein interactions, but charged patches can be equally promiscuous. It will be essential in future work to determine which proteins capable of binding the HIV-1 capsid in vitro are bona fide viral cofactors in cells. Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition MechanismsSummers et al.Cell Host & MicrobeAugust 14, 2019In BriefSummers et al. used a series of protein engineering tools to “trap” the naturally self-polymerizing HIV-1 capsid protein in discrete, soluble fragments. These fragments enabled a precision analysis of the capsid recognition surfaces and binding modes used by host factors to facilitate or block HIV-1 infection. Full-Text PDF Open Archive
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