- Email: [email protected]
A Virus that Can Take the Heat
Structure
Previews A Virus that Can Take the Heat Elin M. Sivertsson1 and Laura S. Itzhaki1,* 1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2014.10.003
Foot-and-mouth disease virus shows remarkable thermal lability, a property that is a particular problem for vaccine preparations. In this issue of Structure, Rinco´n and colleagues show that electrostatic repulsion within the capsid is responsible for this lability, and they present rationally designed mutants with increased thermostability. Foot-and-mouth disease is a highly contagious livestock disease (Grubman and Baxt, 2004). It is endemic in Asia, South America, and Africa and sporadically occurs in other regions, as exemplified by the outbreaks in the UK in 2001 that led to large economic losses. The causal agent of the disease is a picornavirus, foot-and-mouth disease virus (FMDV). Vaccines made from inactivated virus particles are available; however, the low stability of the virus leads to its rapid dissociation with a consequent loss of ability to induce an effective immune response. Virus capsids comprise giant protein assemblies with highly symmetrical architectures. FMDV has an icosahedral capsid, made up of 60 copies each of the proteins VP1–VP3 and the peptide VP4 assembled into 12 pentameric substructures (Acharya et al., 1989). Heat or acid will readily dissociate the capsid into pentamers. In fact, the instability of FMDV at low pH facilitates uncoating of the viral RNA that is contained inside the capsid once the virus reaches the acidic environment in the endosomes of infected cells, and it is thus critical for a successful infectious cycle (Carrillo et al., 1985). However, for the purpose of vaccine production the instability of FMDV is a problem. There has been some success in engineering acid resistance (Ellard et al., 1999; Twomey et al., 1995), but much less in engineering thermal resistance (Ashcroft et al., 2005; Fiedler et al., 2012; Mateo et al., 2008; Porta et al., 2013). Vaccines based on engineered FMDV capsids that can withstand higher temperatures would be very useful, considering the hot climate in many endemic regions where socioeconomic factors preclude adequate cold chain distribution.
A mutant with increased thermal stability, A2065H, was identified in previous studies by the Mateu lab (Mateo et al., 2008). However, the precise mechanism by which the mutation increased capsid stability had not been determined. Could it be that the introduced histidine residue exerted its stabilizing effect by partially neutralizing nearby carboxylates, thus reducing interpentamer electrostatic repulsion? To test this hypothesis, Rinco´n et al. (2014; in this issue of Structure) made a number of acidic-toneutral substitutions (Glu to Gln and Asp to Asn) close to the pentamer interfaces. The authors tested the infectivity of the mutants and found that the majority of the engineered viruses were infectious as well as genetically stable, meaning that the original mutation was preserved after amplification in cells. The infectious virus variants were then tested for thermal stability against dissociation into pentamers. In three of the studied variants, the carboxylate was located in a buried position and participated in several interactions with other residues. As expected, these mutants were destabilized compared to the parent. Upon acidic-to-neutral mutation of six solvent-exposed carboxylates, two variants displayed no change in stability while four mutants (D2068N, E2086Q, D3134N, and D3195N) were significantly stabilized compared to the parent, as shown by their slower dissociation rates at 42 C. Because all of these stable mutants were infectious, it is clear that a thermally labile capsid is not a prerequisite for FMDV infectivity per se. Very strikingly, Rinco´n et al. (2014) next go on to show that calculations based on the very simple Tanford-Kirkwood theoretical model of electrostatics (Tanford and Kirkwood,
1957) are able to predict the destabilizing effects of the residues. Based on their results, Rinco´n et al. (2014) propose a model for FMDV thermostability that can be visualized using an energy diagram, as shown in Figure 1. The instability of the capsid, originating at least in part from intrinsic electrostatic repulsion involving residues D2068, E2086, D3134, and D3195, will lead to ready dissociation into pentamers even at moderate temperatures. Thus, the energy barrier between the associated and dissociated states is very low. By acidic-to-neutral mutations, electrostatic repulsions will be ameliorated. This will decrease the free energy of the assembled (capsid) state, but will not affect the free energy of the transition state where the unfavorable electrostatic interactions have not yet been formed. The net effect will be an increase in the energy barrier between the two states, leading to a reduced rate of irreversible dissociation at a certain temperature; the capsid has been kinetically stabilized. The results of Rinco´n et al. (2014) represent a significant advancement in the field. They show that viral capsid stability can be rationally engineered by changes in electrostatic interactions and that these mutant effects can be predicted using a very simple theoretical model. Not only could this strategy prove beneficial when designing vaccines against various diseases, both traditional vaccines based on inactivated virus and vaccines in the form of recombinant empty capsids, which are often even less thermostable than the nucleic-acid containing capsids (Curry et al., 1997; Porta et al., 2013), but also thermostable capsids could have applications in nanotechnology as molecular containers for the delivery of drugs and other small
Structure 22, November 4, 2014 ª2014 Elsevier Ltd All rights reserved 1549
Structure
Previews REFERENCES
Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D., and Brown, F. (1989). Nature 337, 709–716. Ashcroft, A.E., Lago, H., Macedo, J.M.B., Horn, W.T., Stonehouse, N.J., and Stockley, P.G. (2005). J. Nanosci. Nanotechnol. 5, 2034–2041. Carrillo, E.C., Giachetti, C., and Campos, R. (1985). Virology 147, 118–125. Curry, S., Fry, E., Blakemore, W., Abu-Ghazaleh, R., Jackson, T., King, A., Lea, S., Newman, J., and Stuart, D. (1997). J. Virol. 71, 9743–9752. Ellard, F.M., Drew, J., Blakemore, W.E., Stuart, D.I., and King, A.M.Q. (1999). J. Gen. Virol. 80, 1911–1918. Fiedler, J.D., Higginson, C., Hovlid, M.L., Kislukhin, A.A., Castillejos, A., Manzenrieder, F., Campbell, M.G., Voss, N.R., Potter, C.S., Carragher, B., and Finn, M.G. (2012). Biomacromolecules 13, 2339– 2348. Grubman, M.J., and Baxt, B. (2004). Clin. Microbiol. Rev. 17, 465–493.
Figure 1. Schematic Showing Kinetic Stabilization of FMDV by Mutation The parent virus is in red and the virus with acidic-to-neutral mutation is in blue. The associated state, but not the transition state (z) for irreversible dissociation into pentamers, is stabilized by the mutations, resulting in increased kinetic stability of the capsid.
molecules. More generally, the results provide interesting new insights into how nature has designed giant macromolecular assemblies of incredibly beautiful symmetries that are poised at just the right amount of (in)stability for optimal function. And, for those of us attempting to build biomaterials from self-assembling proteins de novo, it is a
humbling view of the design processes involved. ACKNOWLEDGMENTS E.M.S. acknowledges support from Funds for Women Graduates (FfWG), Lundgren Research Awards, and Cambridge Philosophical Society. L.S.I. acknowledges support from the UK Medical Research Foundation.
Mateo, R., Luna, E., Rinco´n, V., and Mateu, M.G. (2008). J. Virol. 82, 12232–12240. Porta, C., Kotecha, A., Burman, A., Jackson, T., Ren, J., Loureiro, S., Jones, I.M., Fry, E.E., Stuart, D.I., and Charleston, B. (2013). PLoS Pathog. 9, e1003255. Rinco´n, V., Rodrı´guez-Huete, A., Lo´pez-Argu¨ello, S., Ibarra-Molero, B., Sanchez-Ruiz, J.M., Harmsen, M.M., and Mateu, M.G. (2014). Structure 22, this issue, 1560–1570. Tanford, C., and Kirkwood, J.G. (1957). J. Am. Chem. Soc. 79, 5333–5339. Twomey, T., France, L.L., Hassard, S., Burrage, T.G., Newman, J.F.E., and Brown, F. (1995). Virology 206, 69–75.
Chemokine-Receptor Interactions: Solving the Puzzle, Piece by Piece Li Zhang1 and Patricia J. LiWang1,* 1Molecular Cell Biology, University of California, Merced, Merced, CA 95343, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2014.10.004
In an important addition to the chemokine field, Millard and colleagues, in this issue of Structure, report the first structure of a CC chemokine in complex with a sulfated peptide derived from its receptor.
Due to its significant impact on health and disease, the chemokine system has been a target of interest for both academic research and pharmaceutical applications for years. The chemokine
system, encompassing about 50 chemokine proteins that selectively bind to one or several cognate chemokine receptors, forms a sophisticated network that is critical in the mammalian immune
1550 Structure 22, November 4, 2014 ª2014 Elsevier Ltd All rights reserved
system, mediating activation and chemotaxis of leukocytes and playing a role in both homing and inflammation. Dysfunction in the chemokine system has been implicated in health issues
Previews A Virus that Can Take the Heat Elin M. Sivertsson1 and Laura S. Itzhaki1,* 1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2014.10.003
Foot-and-mouth disease virus shows remarkable thermal lability, a property that is a particular problem for vaccine preparations. In this issue of Structure, Rinco´n and colleagues show that electrostatic repulsion within the capsid is responsible for this lability, and they present rationally designed mutants with increased thermostability. Foot-and-mouth disease is a highly contagious livestock disease (Grubman and Baxt, 2004). It is endemic in Asia, South America, and Africa and sporadically occurs in other regions, as exemplified by the outbreaks in the UK in 2001 that led to large economic losses. The causal agent of the disease is a picornavirus, foot-and-mouth disease virus (FMDV). Vaccines made from inactivated virus particles are available; however, the low stability of the virus leads to its rapid dissociation with a consequent loss of ability to induce an effective immune response. Virus capsids comprise giant protein assemblies with highly symmetrical architectures. FMDV has an icosahedral capsid, made up of 60 copies each of the proteins VP1–VP3 and the peptide VP4 assembled into 12 pentameric substructures (Acharya et al., 1989). Heat or acid will readily dissociate the capsid into pentamers. In fact, the instability of FMDV at low pH facilitates uncoating of the viral RNA that is contained inside the capsid once the virus reaches the acidic environment in the endosomes of infected cells, and it is thus critical for a successful infectious cycle (Carrillo et al., 1985). However, for the purpose of vaccine production the instability of FMDV is a problem. There has been some success in engineering acid resistance (Ellard et al., 1999; Twomey et al., 1995), but much less in engineering thermal resistance (Ashcroft et al., 2005; Fiedler et al., 2012; Mateo et al., 2008; Porta et al., 2013). Vaccines based on engineered FMDV capsids that can withstand higher temperatures would be very useful, considering the hot climate in many endemic regions where socioeconomic factors preclude adequate cold chain distribution.
A mutant with increased thermal stability, A2065H, was identified in previous studies by the Mateu lab (Mateo et al., 2008). However, the precise mechanism by which the mutation increased capsid stability had not been determined. Could it be that the introduced histidine residue exerted its stabilizing effect by partially neutralizing nearby carboxylates, thus reducing interpentamer electrostatic repulsion? To test this hypothesis, Rinco´n et al. (2014; in this issue of Structure) made a number of acidic-toneutral substitutions (Glu to Gln and Asp to Asn) close to the pentamer interfaces. The authors tested the infectivity of the mutants and found that the majority of the engineered viruses were infectious as well as genetically stable, meaning that the original mutation was preserved after amplification in cells. The infectious virus variants were then tested for thermal stability against dissociation into pentamers. In three of the studied variants, the carboxylate was located in a buried position and participated in several interactions with other residues. As expected, these mutants were destabilized compared to the parent. Upon acidic-to-neutral mutation of six solvent-exposed carboxylates, two variants displayed no change in stability while four mutants (D2068N, E2086Q, D3134N, and D3195N) were significantly stabilized compared to the parent, as shown by their slower dissociation rates at 42 C. Because all of these stable mutants were infectious, it is clear that a thermally labile capsid is not a prerequisite for FMDV infectivity per se. Very strikingly, Rinco´n et al. (2014) next go on to show that calculations based on the very simple Tanford-Kirkwood theoretical model of electrostatics (Tanford and Kirkwood,
1957) are able to predict the destabilizing effects of the residues. Based on their results, Rinco´n et al. (2014) propose a model for FMDV thermostability that can be visualized using an energy diagram, as shown in Figure 1. The instability of the capsid, originating at least in part from intrinsic electrostatic repulsion involving residues D2068, E2086, D3134, and D3195, will lead to ready dissociation into pentamers even at moderate temperatures. Thus, the energy barrier between the associated and dissociated states is very low. By acidic-to-neutral mutations, electrostatic repulsions will be ameliorated. This will decrease the free energy of the assembled (capsid) state, but will not affect the free energy of the transition state where the unfavorable electrostatic interactions have not yet been formed. The net effect will be an increase in the energy barrier between the two states, leading to a reduced rate of irreversible dissociation at a certain temperature; the capsid has been kinetically stabilized. The results of Rinco´n et al. (2014) represent a significant advancement in the field. They show that viral capsid stability can be rationally engineered by changes in electrostatic interactions and that these mutant effects can be predicted using a very simple theoretical model. Not only could this strategy prove beneficial when designing vaccines against various diseases, both traditional vaccines based on inactivated virus and vaccines in the form of recombinant empty capsids, which are often even less thermostable than the nucleic-acid containing capsids (Curry et al., 1997; Porta et al., 2013), but also thermostable capsids could have applications in nanotechnology as molecular containers for the delivery of drugs and other small
Structure 22, November 4, 2014 ª2014 Elsevier Ltd All rights reserved 1549
Structure
Previews REFERENCES
Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D., and Brown, F. (1989). Nature 337, 709–716. Ashcroft, A.E., Lago, H., Macedo, J.M.B., Horn, W.T., Stonehouse, N.J., and Stockley, P.G. (2005). J. Nanosci. Nanotechnol. 5, 2034–2041. Carrillo, E.C., Giachetti, C., and Campos, R. (1985). Virology 147, 118–125. Curry, S., Fry, E., Blakemore, W., Abu-Ghazaleh, R., Jackson, T., King, A., Lea, S., Newman, J., and Stuart, D. (1997). J. Virol. 71, 9743–9752. Ellard, F.M., Drew, J., Blakemore, W.E., Stuart, D.I., and King, A.M.Q. (1999). J. Gen. Virol. 80, 1911–1918. Fiedler, J.D., Higginson, C., Hovlid, M.L., Kislukhin, A.A., Castillejos, A., Manzenrieder, F., Campbell, M.G., Voss, N.R., Potter, C.S., Carragher, B., and Finn, M.G. (2012). Biomacromolecules 13, 2339– 2348. Grubman, M.J., and Baxt, B. (2004). Clin. Microbiol. Rev. 17, 465–493.
Figure 1. Schematic Showing Kinetic Stabilization of FMDV by Mutation The parent virus is in red and the virus with acidic-to-neutral mutation is in blue. The associated state, but not the transition state (z) for irreversible dissociation into pentamers, is stabilized by the mutations, resulting in increased kinetic stability of the capsid.
molecules. More generally, the results provide interesting new insights into how nature has designed giant macromolecular assemblies of incredibly beautiful symmetries that are poised at just the right amount of (in)stability for optimal function. And, for those of us attempting to build biomaterials from self-assembling proteins de novo, it is a
humbling view of the design processes involved. ACKNOWLEDGMENTS E.M.S. acknowledges support from Funds for Women Graduates (FfWG), Lundgren Research Awards, and Cambridge Philosophical Society. L.S.I. acknowledges support from the UK Medical Research Foundation.
Mateo, R., Luna, E., Rinco´n, V., and Mateu, M.G. (2008). J. Virol. 82, 12232–12240. Porta, C., Kotecha, A., Burman, A., Jackson, T., Ren, J., Loureiro, S., Jones, I.M., Fry, E.E., Stuart, D.I., and Charleston, B. (2013). PLoS Pathog. 9, e1003255. Rinco´n, V., Rodrı´guez-Huete, A., Lo´pez-Argu¨ello, S., Ibarra-Molero, B., Sanchez-Ruiz, J.M., Harmsen, M.M., and Mateu, M.G. (2014). Structure 22, this issue, 1560–1570. Tanford, C., and Kirkwood, J.G. (1957). J. Am. Chem. Soc. 79, 5333–5339. Twomey, T., France, L.L., Hassard, S., Burrage, T.G., Newman, J.F.E., and Brown, F. (1995). Virology 206, 69–75.
Chemokine-Receptor Interactions: Solving the Puzzle, Piece by Piece Li Zhang1 and Patricia J. LiWang1,* 1Molecular Cell Biology, University of California, Merced, Merced, CA 95343, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2014.10.004
In an important addition to the chemokine field, Millard and colleagues, in this issue of Structure, report the first structure of a CC chemokine in complex with a sulfated peptide derived from its receptor.
Due to its significant impact on health and disease, the chemokine system has been a target of interest for both academic research and pharmaceutical applications for years. The chemokine
system, encompassing about 50 chemokine proteins that selectively bind to one or several cognate chemokine receptors, forms a sophisticated network that is critical in the mammalian immune
1550 Structure 22, November 4, 2014 ª2014 Elsevier Ltd All rights reserved
system, mediating activation and chemotaxis of leukocytes and playing a role in both homing and inflammation. Dysfunction in the chemokine system has been implicated in health issues