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Virus assembly, allostery and antivirals

2010; Elsevier BV; Volume: 19; Issue: 1 Linguagem: Inglês

10.1016/j.tim.2010.11.003

1878-4380

Adam Zlotnick, Suchetana Mukhopadhyay,

Viral Infections and Outbreaks Research

Assembly of virus capsids and surface proteins must be regulated to ensure that the resulting complex is an infectious virion. In this review, we examine assembly of virus capsids, focusing on hepatitis B virus and bacteriophage MS2, and formation of glycoproteins in the alphaviruses. These systems are structurally and biochemically well-characterized and are simplest-case paradigms of self-assembly. Published data suggest that capsid and glycoprotein assembly is subject to allosteric regulation, that is regulation at the level of conformational change. The hypothesis that allostery is a common theme in viruses suggests that deregulation of capsid and glycoprotein assembly by small molecule effectors will be an attractive antiviral strategy, as has been demonstrated with hepatitis B virus. Assembly of virus capsids and surface proteins must be regulated to ensure that the resulting complex is an infectious virion. In this review, we examine assembly of virus capsids, focusing on hepatitis B virus and bacteriophage MS2, and formation of glycoproteins in the alphaviruses. These systems are structurally and biochemically well-characterized and are simplest-case paradigms of self-assembly. Published data suggest that capsid and glycoprotein assembly is subject to allosteric regulation, that is regulation at the level of conformational change. The hypothesis that allostery is a common theme in viruses suggests that deregulation of capsid and glycoprotein assembly by small molecule effectors will be an attractive antiviral strategy, as has been demonstrated with hepatitis B virus. The common structural denominator for a typical small virus is a genome surrounded by a shell composed of dozens of copies of the capsid protein. Many small viruses also have lipid envelopes studded with glycoproteins that can facilitate cell entry. Even a small virus is complex, yet its formation is a canonical example of self-assembly. Given the right conditions, the capsid proteins of many viruses will assemble to capsid-like structures, rapidly, in high yield and with high fidelity (Table 1). This observation opens up two distinct fields of study: the process of self-assembly and regulation of assembly. The process of self-assembly is best described by physical chemistry, for example self-assembly can be modeled by a system of differential equations or emulated with small molecules. Regulating assembly, by contrast, is fundamentally biochemical and ultimately biological in nature. Consider our hypothetical virus where unregulated assembly could yield capsids that did not contain the viral genome or toxic accumulation of the fusion protein. These spontaneous assembly reactions must be delayed until the right time and place. Regulation is probably at the level of allostery, which is defined as a conformational change in a molecule, usually a protein, that alters its activity, induced by an effector molecule. In addition to allostery, regulation could require viral or host factors. Regulation of the large event of virus assembly by a small reaction, such as inducing conformational change of a single protein, provides the leverage that controls virus replication in vivo. Disruption of regulation is an ideal target for antiviral therapeutics. Defining the regulation that directly controls the physical chemistry of assembly is a field in its infancy and the focus of this review.Table 1An incomplete list of in vitro icosahedral capsid assembly systemsSpeciesFamilyNumber of proteinsRefsPlant virusesCowpea chlorotic mottle virus, Brome mosaic virusBromo180, 120 and 6071Bancroft J.B. The self-assembly of spherical plant viruses.Adv. Virus Res. 1970; 16: 99-134Crossref PubMed Scopus (216) Google Scholar, 72Bancroft J.B. Hiebert E. Formation of an infectious nucleoprotein from protein and nucleic acid isolated from a small spherical virus.Virology. 1967; 32: 354-356Crossref PubMed Scopus (117) Google ScholarSouthern bean virusSobemo180 and 6073Savithri H.S. Erickson J.W. The self-assembly of the cowpea strain of southern bean mosaic virus: formation of T = 1 and T = 3 nucleoprotein particles.Virology. 1983; 126: 328-335Crossref PubMed Scopus (66) Google ScholarTurnip crinkle virus, Sesbania mosaic virusTombus18074Lokesh G.L. et al.A molecular switch in the capsid protein controls the particle polymorphism in an icosahedral virus.Virology. 2002; 292: 211-223Crossref PubMed Scopus (48) Google ScholarPhysalis mottle virusTymo180 and 6075Sastri M. et al.Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.J. Mol. Biol. 1999; 289: 905-918Crossref PubMed Scopus (26) Google ScholarAnimal virusesHerpes simplex virusHerpes∼200076Newcomb W.W. et al.Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins.J. Virol. 1999; 73: 4239-4250PubMed Google ScholarHepatitis B virusHepadna2406Zlotnick A. et al.A theoretical model successfully identifies features of hepatitis B virus capsid assembly.Biochemistry. 1999; 38: 14644-14652Crossref PubMed Scopus (264) Google ScholarSindbis virus, Ross River virusToga24077Tellinghuisen T.L. et al.In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli.J. Virol. 1999; 73: 5309-5319Crossref PubMed Google Scholar, 78Mukhopadhyay S. et al.In vitro-assembled alphavirus core-like particles maintain a structure similar to that of nucleocapsid cores in mature virus.J. Virol. 2002; 76: 11128-11132Crossref PubMed Scopus (57) Google ScholarHuman papillomavirus 16Papilloma36079Kirnbauer R. et al.Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.J. Virol. 1993; 67: 6929-6936Crossref PubMed Google ScholarPolyomavirus SV40Polyoma36080Salunke D.M. et al.Self-assembly of purified polyomavirus capsid protein VP1.Cell. 1986; 46: 895-904Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 81Colomar M.C. et al.Opening and refolding of simian virus 40 and in vitro packaging of foreign DNA.J. Virol. 1993; 67: 2779-2786Crossref PubMed Google ScholarPoliovirus, Foot and mouth disease virusPicorna18082Rombaut B. et al.In vitro assembly of poliovirus empty capsids: antigenic consequences and immunological assay of the morphopoietic factor.Virology. 1984; 135: 546-550Crossref PubMed Scopus (17) Google Scholar, 83Goodwin S. et al.Foot-and-mouth disease virus assembly: processing of recombinant capsid precursor by exogenous protease induces self-assembly of pentamers in vitro in a myristoylation-dependent manner.J. Virol. 2009; 83: 11275-11282Crossref PubMed Scopus (39) Google ScholarRous sarcoma virus HIVRetro∼200022Campbell S. Rein A. In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain.J. Virol. 1999; 73: 2270-2279Crossref PubMed Google Scholar, 23Campbell S. Vogt V.M. Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1.J. Virol. 1995; 69: 6487-6497Crossref PubMed Google ScholarHepatitis E virusHepe18084Li T.C. et al.Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus.J. Virol. 2005; 79: 12999-13006Crossref PubMed Scopus (113) Google ScholarBacteriophagesHK97, LambdaSipho42012Hendrix R.W. Duda R.L. Bacteriophage HK97 head assembly: a protein ballet.Adv. Virus Res. 1998; 50: 235-288Crossref PubMed Scopus (76) Google Scholar, 85Gaussier H. et al.Building a virus from scratch: assembly of an infectious virus using purified components in a rigorously defined biochemical assay system.J. Mol. Biol. 2006; 357: 1154-1166Crossref PubMed Scopus (46) Google ScholarP22Podo42013Prevelige P.E. et al.Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells.Biophys. J. 1993; 64: 824-835Abstract Full Text PDF PubMed Scopus (224) Google ScholarMS2, R17Levi18033Beckett D. et al.Roles of operator and non-operator RNA sequences in bacteriophage R17 capsid assembly.J. Mol. Biol. 1988; 204: 939-947Crossref PubMed Scopus (88) Google Scholar, 35Stockley P.G. et al.A simple, RNA-mediated allosteric switch controls the pathway to formation of a T = 3 viral capsid.J. Mol. Biol. 2007; 369: 541-552Crossref PubMed Scopus (111) Google ScholarøX174Micro30016Fane B.A. Prevelige Jr., P.E. Mechanism of scaffolding-assisted viral assembly.in: Chiu W. Johnson J.E. Virus Structure. Academic Press, 2003: 259-299Crossref Scopus (102) Google Scholar Open table in a new tab In this review, we will separately examine the assembly of virus capsids and the membrane-bound glycoprotein complexes present on the exterior of enveloped viruses. Both the capsid and glycoprotein are protein oligomers; from a physical–chemical perspective, their assembly is similar; from a biological perspective they demonstrably have similar elements of regulation. Although there are numerous experimental systems (Table 1, Table 2), the discussion of capsid assembly will focus on hepatitis B virus (HBV) with significant references to retroviruses and bacteriophage MS2. Furthermore, small molecules that dramatically affect HBV assembly have demonstrable antiviral activity. The discussion on glycoprotein assembly will focus on the formation of the spike complex of alphaviruses as the spike is composed of two proteins which intricately interact and are required for viral entry. Finally, we will discuss potential steps in assembly that could be targeted for designing antiviral therapeutics.Table 2An incomplete list of enveloped virus structuresaThe structures of most enveloped viruses, either entire particles or portions of the particle, have been determined using cryo-electron microscopy either by single-particle averaging or tomography.ExampleFamilyRefsMethod(s)Viruses with an icosahedral core and icosahedral glycoprotein layerSindbis virus, Venezuelan equine encephalitis virusToga51Mukhopadhyay S. et al.Mapping the structure and function of the E1 and E2 glycoproteins in alphaviruses.Structure. 2006; 14: 63-73Abstract Full Text Full Text PDF PubMed Scopus (168) Google ScholarSingle-particle averagingViruses with an icosahedral core and local symmetry in the glycoprotein layerHepatitis B virusHepadna86Dryden K.A. et al.Native hepatitis B virions and capsids visualized by electron cryomicroscopy.Mol. Cell. 2006; 22: 843-850Abstract Full Text Full Text PDF PubMed Scopus (138) Google ScholarSingle-particle averagingTula virusBunya87Huiskonen J.T. et al.Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses.J. Virol. 2010; 84: 4889-4897Crossref PubMed Scopus (112) Google ScholarTomographyViruses with an icosahedral core and no symmetry in the glycoprotein layerEpstein–Barr virus, herpes virusHerpes88Grunewald K. et al.Three-dimensional structure of herpes simplex virus from cryo-electron tomography.Science. 2003; 302: 1396-1398Crossref PubMed Scopus (469) Google ScholarTomographyViruses with local symmetry in the core and no symmetry the glycoprotein layerSimian immunodeficiency virus, HIV, murine leukemia virusRetro31Briggs J.A. et al.Structure and assembly of immature HIV.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 11090-11095Crossref PubMed Scopus (297) Google Scholar, 89Forster F. et al.Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4729-4734Crossref PubMed Scopus (246) Google ScholarTomographyViruses with no symmetry in the core but icosahedral glycoprotein layerDengue virus, West Nile virus, tick-borne encephalitis virusFlavi90Zhang W. et al.Visualization of membrane protein domains by cryo-electron microscopy of dengue virus.Nat. Struct. Biol. 2003; 10: 907-912Crossref PubMed Scopus (374) Google ScholarSingle-particle averagingRift Valley fever virus, Uukuniemi virusBunya91Freiberg A.N. et al.Three-dimensional organization of Rift Valley fever virus revealed by cryoelectron tomography.J. Virol. 2008; 82: 10341-10348Crossref PubMed Scopus (108) Google ScholarTomographyViruses with helical symmetry in core and glycoprotein symmetry undeterminedVesicular stomatitis virus, measles virusRhabdo92Ge, P. et al. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science 327, 689–693Google ScholarSingle-particle averagingViruses with no symmetry observed in either the core nor the glycoprotein layerMouse hepatitis virus, severe acute respiratory syndrome virusCorona93Beniac D.R. et al.Architecture of the SARS coronavirus prefusion spike.Nat. Struct. Mol. Biol. 2006; 13: 751-752Crossref PubMed Scopus (231) Google ScholarSingle-particle averagingVaccinia virusPox94Cyrklaff M. et al.Cryo-electron tomography of vaccinia virus.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2772-2777Crossref PubMed Scopus (167) Google ScholarTomographyInfluenza virusOrthomyxo95Harris A. et al.Influenza virus pleiomorphy characterized by cryoelectron tomography.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19123-19127Crossref PubMed Scopus (384) Google ScholarTomographyEbola virusFilo96Welsch, S. et al. Electron tomography reveals the steps in filovirus budding. PLoS Pathog. 6, e1000875Google ScholarTomographyParainfluenza virus, Hendra virus, Sendai virusParamyxo97Loney C. et al.Paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus.J. Virol. 2009; 83: 8191-8197Crossref PubMed Scopus (66) Google ScholarTomographya The structures of most enveloped viruses, either entire particles or portions of the particle, have been determined using cryo-electron microscopy either by single-particle averaging or tomography. Open table in a new tab The capsids of icosahedral viruses have tens to hundreds of copies of the capsid protein(s). The simplest vision of virus capsid assembly is one where rigid assembly units (AUs) collide by Brownian motion, interact with perfect geometry and associate irreversibly [1Porterfield J.Z. Zlotnick A. An overview of capsid assembly kinetics.in: Stockley P.G. Twarock R. Emerging Topics in Physical Virology. Imperial College Press, 2010: 131-158Crossref Scopus (11) Google Scholar]. Like any utopia, this vision fails examination: assembly simulations are incredibly sensitive to kinetic traps consisting of partial capsids that have a negligible chance of completion due to depletion of subunits (Figure 1). Instead, assembly simulations, from master equations that treat assembly reactions as a well-mixed solution [2Endres D. Zlotnick A. Model-based analysis of assembly kinetics for virus capsids or other spherical polymers.Biophys. J. 2002; 83: 1217-1230Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 3Keef T. et al.Master equation approach to the assembly of viral capsids.J. Theor. Biol. 2006; 242: 713-721Crossref PubMed Scopus (51) Google Scholar] to molecular dynamics that describe stochastic formation of single capsids [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar, 5Rapaport D.C. Role of reversibility in viral capsid growth: a paradigm for self-assembly.Phys. Rev. Lett. 2008; 101: 186101Crossref PubMed Scopus (119) Google Scholar], concur on three generalizations (Box 1). First, weak interactions are necessary to minimize errors and kinetic traps. Weak interactions also contribute to a defined nucleation step. Second, nucleation minimizes the initiation of assembly, decreasing kinetic trapping of intermediates due to depletion of AUs. Third, the initial kinetic phase where there is little capsid formation is due to the time required to build the steady state of intermediates that supports subsequent assembly. More sophisticated mathematical models that incorporate the biological details of individual viruses (e.g. nucleic acid, scaffolding and allostery) will help to identify opportunities to interfere with assembly.Box 1An analytical description of capsid assemblyProbably the simplest way to describe assembly of a capsid of N assembly units (AUs) is as a progressive series of intermediates [100Zlotnick A. To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes.J. Mol. Biol. 1994; 241: 59-67Crossref PubMed Scopus (278) Google Scholar]. To quantify this description, one can assume that all contacts between AUs have the same energy, energies are additive, microscopic forward rates are identical (subject to statistical considerations) and that backward rates are the product of forward rates, dissociation constants and statistical terms. The result is a series of rate equations each with terms for assembly and disassembly. Two equations in the series are unique: the monomer equation references all intermediates and the final equation is a dead-end with only one build-up and one build-down term.d[n-mer]/dt=kforward,n[(n−1)mer][monomer]−kforward,n+1[n-mer][monomer]−kbackward,n[n-mer]+kbackward,n+1[(n+1)mer]Incorporating weak interactions between AUs and a slow nucleation step to limit the initiation of assembly results in robust reactions with minimal kinetics traps where errors and (meta)stable intermediates accumulate [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar, 39Moisant P. et al.Exploring the paths of (virus) assembly.Biophys. J. 2010; 99: 1350-1357Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. This model necessarily leads to sigmoidal kinetics and, at equilibrium, a pseudocritical concentration of free AU [5Rapaport D.C. Role of reversibility in viral capsid growth: a paradigm for self-assembly.Phys. Rev. Lett. 2008; 101: 186101Crossref PubMed Scopus (119) Google Scholar, 100Zlotnick A. To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes.J. Mol. Biol. 1994; 241: 59-67Crossref PubMed Scopus (278) Google Scholar]. The resulting model recapitulates most of the features observed in vitro (Figure I).Assembly can be described in much greater detail as stochastic reactions using discrete event simulators or coarse-grained molecular dynamics [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar]. These molecular simulations provide detail, showing conditions where association of intermediates could be an important path, where reversibility is crucial [5Rapaport D.C. Role of reversibility in viral capsid growth: a paradigm for self-assembly.Phys. Rev. Lett. 2008; 101: 186101Crossref PubMed Scopus (119) Google Scholar], and how subunit geometry can affect the size and shape of assembly products [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar]. This statistical mechanical view of assembly leads to a view that is fundamentally similar to the thermodynamic–kinetic view described above [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar]. However, the detail provides additional insights into the reactions and the behaviors that can be anticipated by biological molecules. Probably the simplest way to describe assembly of a capsid of N assembly units (AUs) is as a progressive series of intermediates [100Zlotnick A. To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes.J. Mol. Biol. 1994; 241: 59-67Crossref PubMed Scopus (278) Google Scholar]. To quantify this description, one can assume that all contacts between AUs have the same energy, energies are additive, microscopic forward rates are identical (subject to statistical considerations) and that backward rates are the product of forward rates, dissociation constants and statistical terms. The result is a series of rate equations each with terms for assembly and disassembly. Two equations in the series are unique: the monomer equation references all intermediates and the final equation is a dead-end with only one build-up and one build-down term.d[n-mer]/dt=kforward,n[(n−1)mer][monomer]−kforward,n+1[n-mer][monomer]−kbackward,n[n-mer]+kbackward,n+1[(n+1)mer] Incorporating weak interactions between AUs and a slow nucleation step to limit the initiation of assembly results in robust reactions with minimal kinetics traps where errors and (meta)stable intermediates accumulate [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar, 39Moisant P. et al.Exploring the paths of (virus) assembly.Biophys. J. 2010; 99: 1350-1357Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. This model necessarily leads to sigmoidal kinetics and, at equilibrium, a pseudocritical concentration of free AU [5Rapaport D.C. Role of reversibility in viral capsid growth: a paradigm for self-assembly.Phys. Rev. Lett. 2008; 101: 186101Crossref PubMed Scopus (119) Google Scholar, 100Zlotnick A. To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes.J. Mol. Biol. 1994; 241: 59-67Crossref PubMed Scopus (278) Google Scholar]. The resulting model recapitulates most of the features observed in vitro (Figure I). Assembly can be described in much greater detail as stochastic reactions using discrete event simulators or coarse-grained molecular dynamics [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar]. These molecular simulations provide detail, showing conditions where association of intermediates could be an important path, where reversibility is crucial [5Rapaport D.C. Role of reversibility in viral capsid growth: a paradigm for self-assembly.Phys. Rev. Lett. 2008; 101: 186101Crossref PubMed Scopus (119) Google Scholar], and how subunit geometry can affect the size and shape of assembly products [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar]. This statistical mechanical view of assembly leads to a view that is fundamentally similar to the thermodynamic–kinetic view described above [4Elrad O.M. Hagan M.F. Mechanisms of size control and polymorphism in viral capsid assembly.Nano Lett. 2008; 8: 3850-3857Crossref PubMed Scopus (72) Google Scholar]. However, the detail provides additional insights into the reactions and the behaviors that can be anticipated by biological molecules. Experimental observation of assembly of empty HBV capsids is in good agreement with the predictions of theoretical models [6Zlotnick A. et al.A theoretical model successfully identifies features of hepatitis B virus capsid assembly.Biochemistry. 1999; 38: 14644-14652Crossref PubMed Scopus (264) Google Scholar] (Box 1). HBV has a T = 4 icosahedral capsid composed of 120 AUs, the homodimeric core protein (Cp) [7Seeger C. et al.Hepadnaviruses.in: Knipe D.M. Fields Virology. Lippincott Williams and Wilkins, 2007: 2977-3029Google Scholar]. Cp can be assembled in vitro in response to ionic strength [6Zlotnick A. et al.A theoretical model successfully identifies features of hepatitis B virus capsid assembly.Biochemistry. 1999; 38: 14644-14652Crossref PubMed Scopus (264) Google Scholar] and the kinetics are sigmoidal [6Zlotnick A. et al.A theoretical model successfully identifies features of hepatitis B virus capsid assembly.Biochemistry. 1999; 38: 14644-14652Crossref PubMed Scopus (264) Google Scholar]. The average association energy between two subunits is –3 to –4 kcal/mol, corresponding to a millimolar dissociation constant. Because subunits are tetravalent, the weak association energy corresponds to a micromolar pseudocritical concentration [8Ceres P. Zlotnick A. Weak protein–protein interactions are sufficient to drive assembly of hepatitis B virus capsids.Biochemistry. 2002; 41: 11525-11531Crossref PubMed Scopus (312) Google Scholar]. In vitro HBV assembly is resistant to, but not altogether immune to, kinetic traps [9Stray S.J. et al.A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8138-8143Crossref PubMed Scopus (233) Google Scholar, 10Stray S.J. et al.Zinc ions trigger conformational change and oligomerization of hepatitis B virus capsid protein.Biochemistry. 2004; 43: 9989-9998Crossref PubMed Scopus (64) Google Scholar]. HBV is a simplest-case system, a homopolymer of dimeric AUs. In cowpea chlorotic mottle virus and bacteriophage HK97, different AU oligomers participate in assembly [11Zlotnick A. et al.Mechanism of capsid assembly for an icosahedral plant virus.Virology. 2000; 277: 450-456Crossref PubMed Scopus (256) Google Scholar, 12Hendrix R.W. Duda R.L. Bacteriophage HK97 head assembly: a protein ballet.Adv. Virus Res. 1998; 50: 235-288Crossref PubMed Scopus (76) Google Scholar]. In many viruses, scaffold proteins support assembly. Bacteriophage P22 has a scaffold protein that thermodynamically and kinetically contributes to assembly [13Prevelige P.E. et al.Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells.Biophys. J. 1993; 64: 824-835Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 14Parent K.N. et al.Electrostatic interactions govern both nucleation and elongation during phage P22 procapsid assembly.Virology. 2005; 340: 33-45Crossref PubMed Scopus (49) Google Scholar]. Excess P22 scaffold can actually block assembly by trapping numerous intermediates [14Parent K.N. et al.Electrostatic interactions govern both nucleation and elongation during phage P22 procapsid assembly.Virology. 2005; 340: 33-45Crossref PubMed Scopus (49) Google Scholar]. Scaffolds can play roles in switching morphologies, as in bacteriophages P2 and P4 [15Wang S. et al.Assembly of bacteriophage P2 and P4 procapsids with internal scaffolding protein.Virology. 2006; 348: 133-140Crossref PubMed Scopus (29) Google Scholar], and complex roles in subsequent maturation [16Fane B.A. Prevelige Jr., P.E. Mechanism of scaffolding-assisted viral assembly.in: Chiu W. Johnson J.E. Virus Structure. Academic Press, 2003: 259-299Crossref Scopus (102) Google Scholar]. Thus, scaffold proteins could direct geometry, stability and a measure of regulation by imposing stepwise assembly. Viral nucleic acid can also serve as a molecular scaffold. Viral RNA has been considered as an 'antenna' to attract free AUs [17Datta S.A. et al.Conformation of the HIV-1 Gag protein in solution.J. Mol. Biol. 2007; 365: 812-824Crossref PubMed Scopus (111) Google Scholar] and as a platform that attracts and organizes AUs on its surface [18Hagan M.F. A theory for viral capsid assembly around electrostatic cores.J. Chem. Phys. 2009; 130: 114902Crossref PubMed Scopus (64) Google Scholar]. Assembly driven by RNA can be thought of in terms of the McGhee–von Hippel model of nonspecific protein binding to a surface [19McGhee J.D. von Hippel P.H. Theoretical aspects of DNA–protein interactions: cooperative and non-cooperative binding of large ligands to a one dimensional homogeneous lattice.J. Mol. Biol. 1974; 86: 469-489Crossref PubMed Scopus (2826) Google Scholar], which considers that the association for nucleic acid (NA), KNA, is modified by a cooperativity coefficient, ω, based on the protein–protein association constant. An ω of 1 indicates no cooperativity; an ω >1000 results in steep cooperativity and reactions that appear to be two-state. Cowpea chlorotic mottle virus, which does not assemble under RNA-binding conditions, binds RNA with low cooperativity, displaying gradual assembly and partial capsids [20Johnson J.M. et al.Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus.J. Mol. Biol. 2004; 335: 455-464Crossref PubMed Scopus (76) Google Scholar]. HBV, where the reciprocal of the pseudocritical concentration (equivalent to ω) is ∼105 under physiological conditions, binds RNA tenaciously and with high cooperativity, resulting in quantitative assembly under mild conditions [21Porterfield J.Z. et al.Full-length HBV core protein packages viral and heterologous RNA with similar high cooperativity.J. Virol. 2010; 84: 7174-7184Crossref PubMed Scopus (128) Google Scholar]. A NA scaffold can thus concentrate the capsid protein and provide additional association energy. Simple theoretical models fail to describe experimental results where induced conformational changes activate assembly. This behavior fits the definition of allostery. NA-regulated allostery is observed with bacteriophage MS2 and retroviruses, for example human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Retroviral Gag, the 'capsid protein' of immature retroviruses, is a multidomain protein. The elements of Gag that are most important for this discussion are the two-domain capsid (CA) segment (the primary mediator of Gag–Gag interactions), a spacer peptide that follows CA, and the RNA-binding nucleocapsid (NC) segment. The earliest suggestion of allostery in retrovirus assembly came from in vitro assembly studies using DNA oligomers and Gag [22Campbell S. Rein A. In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain.J. Virol. 1999; 73: 2270-