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Raising the Curtain on the Structure of Luteovirids
Structure
Previews Raising the Curtain on the Structure of Luteovirids John E. Johnson1,* 1Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.11.008
Luteovirids rank among the most destructive viruses of economically important crops. Until now their structures have only been inferred by inadequate homology models due to their phloem-limited infection and inadequate yields. Employing virus-like particles, Byrne et al. (2019) now report near-atomic resolution structures of two family members providing important functional insights.
In this issue of Structure, Ranson, Lomonossoff, and colleagues (Byrne et al., 2019) present the first structures of members of the luteovirids. The structures result from the use of a novel plant-based protein expression system (Peyret and Lomonossoff, 2013) that allowed the formation of virus-like particles (VLPs) of the luteovirus barley yellow dwarf virus (BYDV) and the polerovirus potato leafroll virus (PLRV) when the respective capsid proteins were expressed with this system in the plant N. benthamiana. Historically, there has been very limited structural information for these viruses since infection of plants is complicated by a required aphid interaction to make the particles infectious and luteoviruses replicate only in the plant vasculature and not in the cytoplasm of leaf cells like many plant viruses. In spite of the small amount of virus available from infected plants, the damage is devastating to economically important crops. Because of their economic importance, both of these viruses have been studied for over 60 years with a great deal of knowledge gained about their gene expression. Like many RNA plant viruses, the luteoviruses utilize the genomic RNA as a message for translation with statistically robust (5%) readthrough events of stop codons and 1 frameshifts, which are facilitated by cis-acting RNA sequences, to generate unique proteins (Dreher and Miller, 2006). A subgenomic RNA, with both 50 and internal start sites in different reading frames, is the message for the capsid protein in BYDV and PLRV. Initiating at the 50 end of the subgenome, the capsid proteins are produced predominantly as the protein observed in the structures reported; however, about 5% of the time the ribosome reads through the stop codon and
produces a protein with an additional 450 residues. The readthrough domain (RTD) is on the surface of the particle of closely related viruses and shown to be functionally important during the aphid stage of virus particle transmission (Brault et al., 1995). The expression system for producing the luteovirid VLPs is based on the genome of another ssRNA plant virus, cowpea mosaic virus (CPMV). Authentic CPMV is highly amenable to surface mutations and has been an excellent tool for virus-based nanotechnology for 30 years, serving as a platform for the attachment of many different biological and chemical entities producing novel functions (Steinmetz et al., 2009). Recently, authentic CPMV has been shown to be a powerful suppressor of oncogenic tumors (Murray et al., 2019). Through a remarkable series of developments, described in detail (Peyret and Lomonossoff, 2013), a CPMV-based transient expression system was advanced to where the gene for the protein of choice is straightforwardly placed into the highly sophisticated cassette shown in Byrne et al. (2019). The entire vector is then placed directly into N. benthamiana in the context of the Agrobacterium tumefaciens-mediated delivery system where extraordinary quantities of readily purified protein are produced by highly efficient expression of the mRNA and translation of the gene placed in the cassette. In the case of viral capsid proteins, the gene products frequently assemble into VLPs that are structurally closely similar to the authentic capsids and have been used as vaccines, platforms in biotechnology, and plant virus diagnostics. Based on the particle size (280 A˚) estimated from negative stained electron
microscopy (EM), the size of the major capsid proteins (25 kD), and the mass of the ssRNA genome (5–6 kb) in these viruses it has been assumed that they have T = 3 quasi symmetry with 180 subunits as observed in many other ssRNA plant viruses with comparably sized components. The VLPs in this study by the Ranson group (Byrne et al., 2019) were employed to produce near-atomic resolution structures at 3 A˚ (BYDV) and 3.4 A˚ (PLRV) with cryoelectron microscopy and image reconstruction (cryo-EM). The structures have canonical T = 3 capsids formed by 180 subunits. The viral jelly roll folds observed in the subunits are of minimal size (140 residues; the smallest jelly roll in the viperdb database; Ho et al., 2018), with short connectors between strands of the beta sheets and two short helices that appear in all jelly roll structures determined (Figures 1A and 1B). The quaternary structure environments and differences in tertiary structures observed to accommodate them display classic quasi-equivalence (Figure 1C) with small adjustments in loops forming 5-fold and quasi 6-fold interactions. Byrne et al. (2019) mapped mutations, previously shown to effect aphid transmissibility (Kaplan et al., 2007), onto their structure of PLRV and demonstrated their surface accessibility. They also showed the shortcomings of previous homology modeling of these residues in that it failed to accurately predict the locations of the changes on the authentic subunit structure determined. There is a potential conundrum regarding the roles of the RTD and the capsid protein in determining aphid interactions. The role of the RTD in aphid transmission was previously established (Brault et al., 1995), but a more recent study identifying the aphid gut
Structure 27, December 3, 2019 ª 2019 Elsevier Ltd. 1735
Structure
Previews
Angstrom, but the VLPs were easily distinguishable from authentic virus in that particle proteolysis occurred at a rate 73 faster than that of authentic virus (Bothner et al., 1999). Thus, solution methods to explore the properties of VLPs and to compare them with virus are valuable to execute when possible.
REFERENCES Bothner, B., Schneemann, A., Marshall, D., Reddy, V., Johnson, J.E., and Siuzdak, G. (1999). Crystallographically identical virus capsids display different properties in solution. Nat. Struct. Biol. 6, 114–116. Brault, V., van den Heuvel, J.F., Verbeek, M., Ziegler-Graff, V., Reutenauer, A., Herrbach, E., Garaud, J.C., Guilley, H., Richards, K., and Jonard, G. (1995). Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74. EMBO J. 14, 650–659.
Figure 1. Cartoon Representations of the Jelly Roll and Particle Structures Associated with Luteovirids (A) The virus jelly roll has a simple topology with two sets of four beta strands arranged in a long loop. The color of the strands indicates the sheets they form when the linear structure is twisted about the short loops (as in making a bakery jelly roll) to create the tertiary structure. (B) The jelly roll tertiary structure demonstrating an idealized trapezoidal shape with adjacent symmetry elements (the pentamer is icosahedral and the trimer and dimer axes are quasi-symmetry axes) that create the quaternary structure in (C). Strand color coding corresponds to (A). (C) A rhombic triacontahedron representing the quaternary structure in the T = 3 luteovirids. The icosahedral asymmetric unit is formed by three subunits (A, B, C) with identical sequences but different environments. Icosahedral symmetry elements are shown in yellow; quasi-symmetry operators are shown in white.
receptor for luteovirus pea enation mosaic virus (PEMV) as alanyl aminopeptidase N (APN) (Linz et al., 2015) used labels on the virus capsid protein (sans RTD) and labels with the RTD domain to determine affinity and location of the receptor. Consistent with the mutants mapped by Byrne et al. (2019), more than 303 coat protein (CP)-label bound to the aphid APN receptor compared to RTD-label. It may be that there are different protein functionalities (CP and CP-RTD) for different receptors as the virus circulates through the aphid. Production of icosahedral VLPs for all the luteovirids investigated was not entirely successful. PEMV capsid protein was expressed in the same manner as BYDV and PLRV, but the particles formed were pleomorphic, lacking icosahedral symmetry. Well-controlled homol-
1736 Structure 27, December 3, 2019
ogy modeling, based on the closely similar sequences of PEMV to BYDV and PLRV, was performed to generate reliable tertiary and quaternary structures for this virus capsid, completing the structures for the three viruses. The use of VLPs for structural models of authentic viruses is now well established and their usefulness is exceptionally important, especially in cases like the luteovirids where sufficient quantities of material are not available for biophysical studies. However, it must be noted that while they are excellent models for structure, the authentic virus particles can have properties different from the VLPs that are important for their biology. A case in point is flock house virus where VLPs crystallized isomorphous with authentic virus and the crystal structures had root mean square deviations of well below an
Byrne, M.J., Steele, J.F.C., Hesketh, E.L., Walden, M., Thompson, R.F., Lomonossoff, G.P., and Ranson, N.A. (2019). Combining Transient Expression and Cryo-EM to Obtain HighResolution Structures of Luteovirid Particles. Structure 27, this issue, 1761–1770. Dreher, T.W., and Miller, W.A. (2006). Translational control in positive strand RNA plant viruses. Virology 344, 185–197. Ho, P.T., Montiel-Garcia, D.J., Wong, J.J., CarrilloTripp, M., Brooks, C.L., 3rd, Johnson, J.E., and Reddy, V.S. (2018). VIPERdb: A Tool for Virus Research. Annu. Rev. Virol. 5, 477–488. Kaplan, I.B., Lee, L., Ripoll, D.R., Palukaitis, P., Gildow, F., and Gray, S.M. (2007). Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission. J. Gen. Virol. 88, 1821–1830. Linz, L.B., Liu, S., Chougule, N.P., and Bonning, B.C. (2015). In Vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector. J. Virol. 89, 11203–11212. Murray, A.A., Sheen, M.R., Veliz, F.A., Fiering, S.N., and Steinmetz, N.F. (2019). In Situ Vaccination of Tumors Using Plant Viral Nanoparticles. Methods Mol. Biol. 2000, 111–124. Peyret, H., and Lomonossoff, G.P. (2013). The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant Mol. Biol. 83, 51–58. Steinmetz, N.F., Lin, T., Lomonossoff, G.P., and Johnson, J.E. (2009). Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. Curr. Top. Microbiol. Immunol. 327, 23–58.
Previews Raising the Curtain on the Structure of Luteovirids John E. Johnson1,* 1Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.11.008
Luteovirids rank among the most destructive viruses of economically important crops. Until now their structures have only been inferred by inadequate homology models due to their phloem-limited infection and inadequate yields. Employing virus-like particles, Byrne et al. (2019) now report near-atomic resolution structures of two family members providing important functional insights.
In this issue of Structure, Ranson, Lomonossoff, and colleagues (Byrne et al., 2019) present the first structures of members of the luteovirids. The structures result from the use of a novel plant-based protein expression system (Peyret and Lomonossoff, 2013) that allowed the formation of virus-like particles (VLPs) of the luteovirus barley yellow dwarf virus (BYDV) and the polerovirus potato leafroll virus (PLRV) when the respective capsid proteins were expressed with this system in the plant N. benthamiana. Historically, there has been very limited structural information for these viruses since infection of plants is complicated by a required aphid interaction to make the particles infectious and luteoviruses replicate only in the plant vasculature and not in the cytoplasm of leaf cells like many plant viruses. In spite of the small amount of virus available from infected plants, the damage is devastating to economically important crops. Because of their economic importance, both of these viruses have been studied for over 60 years with a great deal of knowledge gained about their gene expression. Like many RNA plant viruses, the luteoviruses utilize the genomic RNA as a message for translation with statistically robust (5%) readthrough events of stop codons and 1 frameshifts, which are facilitated by cis-acting RNA sequences, to generate unique proteins (Dreher and Miller, 2006). A subgenomic RNA, with both 50 and internal start sites in different reading frames, is the message for the capsid protein in BYDV and PLRV. Initiating at the 50 end of the subgenome, the capsid proteins are produced predominantly as the protein observed in the structures reported; however, about 5% of the time the ribosome reads through the stop codon and
produces a protein with an additional 450 residues. The readthrough domain (RTD) is on the surface of the particle of closely related viruses and shown to be functionally important during the aphid stage of virus particle transmission (Brault et al., 1995). The expression system for producing the luteovirid VLPs is based on the genome of another ssRNA plant virus, cowpea mosaic virus (CPMV). Authentic CPMV is highly amenable to surface mutations and has been an excellent tool for virus-based nanotechnology for 30 years, serving as a platform for the attachment of many different biological and chemical entities producing novel functions (Steinmetz et al., 2009). Recently, authentic CPMV has been shown to be a powerful suppressor of oncogenic tumors (Murray et al., 2019). Through a remarkable series of developments, described in detail (Peyret and Lomonossoff, 2013), a CPMV-based transient expression system was advanced to where the gene for the protein of choice is straightforwardly placed into the highly sophisticated cassette shown in Byrne et al. (2019). The entire vector is then placed directly into N. benthamiana in the context of the Agrobacterium tumefaciens-mediated delivery system where extraordinary quantities of readily purified protein are produced by highly efficient expression of the mRNA and translation of the gene placed in the cassette. In the case of viral capsid proteins, the gene products frequently assemble into VLPs that are structurally closely similar to the authentic capsids and have been used as vaccines, platforms in biotechnology, and plant virus diagnostics. Based on the particle size (280 A˚) estimated from negative stained electron
microscopy (EM), the size of the major capsid proteins (25 kD), and the mass of the ssRNA genome (5–6 kb) in these viruses it has been assumed that they have T = 3 quasi symmetry with 180 subunits as observed in many other ssRNA plant viruses with comparably sized components. The VLPs in this study by the Ranson group (Byrne et al., 2019) were employed to produce near-atomic resolution structures at 3 A˚ (BYDV) and 3.4 A˚ (PLRV) with cryoelectron microscopy and image reconstruction (cryo-EM). The structures have canonical T = 3 capsids formed by 180 subunits. The viral jelly roll folds observed in the subunits are of minimal size (140 residues; the smallest jelly roll in the viperdb database; Ho et al., 2018), with short connectors between strands of the beta sheets and two short helices that appear in all jelly roll structures determined (Figures 1A and 1B). The quaternary structure environments and differences in tertiary structures observed to accommodate them display classic quasi-equivalence (Figure 1C) with small adjustments in loops forming 5-fold and quasi 6-fold interactions. Byrne et al. (2019) mapped mutations, previously shown to effect aphid transmissibility (Kaplan et al., 2007), onto their structure of PLRV and demonstrated their surface accessibility. They also showed the shortcomings of previous homology modeling of these residues in that it failed to accurately predict the locations of the changes on the authentic subunit structure determined. There is a potential conundrum regarding the roles of the RTD and the capsid protein in determining aphid interactions. The role of the RTD in aphid transmission was previously established (Brault et al., 1995), but a more recent study identifying the aphid gut
Structure 27, December 3, 2019 ª 2019 Elsevier Ltd. 1735
Structure
Previews
Angstrom, but the VLPs were easily distinguishable from authentic virus in that particle proteolysis occurred at a rate 73 faster than that of authentic virus (Bothner et al., 1999). Thus, solution methods to explore the properties of VLPs and to compare them with virus are valuable to execute when possible.
REFERENCES Bothner, B., Schneemann, A., Marshall, D., Reddy, V., Johnson, J.E., and Siuzdak, G. (1999). Crystallographically identical virus capsids display different properties in solution. Nat. Struct. Biol. 6, 114–116. Brault, V., van den Heuvel, J.F., Verbeek, M., Ziegler-Graff, V., Reutenauer, A., Herrbach, E., Garaud, J.C., Guilley, H., Richards, K., and Jonard, G. (1995). Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74. EMBO J. 14, 650–659.
Figure 1. Cartoon Representations of the Jelly Roll and Particle Structures Associated with Luteovirids (A) The virus jelly roll has a simple topology with two sets of four beta strands arranged in a long loop. The color of the strands indicates the sheets they form when the linear structure is twisted about the short loops (as in making a bakery jelly roll) to create the tertiary structure. (B) The jelly roll tertiary structure demonstrating an idealized trapezoidal shape with adjacent symmetry elements (the pentamer is icosahedral and the trimer and dimer axes are quasi-symmetry axes) that create the quaternary structure in (C). Strand color coding corresponds to (A). (C) A rhombic triacontahedron representing the quaternary structure in the T = 3 luteovirids. The icosahedral asymmetric unit is formed by three subunits (A, B, C) with identical sequences but different environments. Icosahedral symmetry elements are shown in yellow; quasi-symmetry operators are shown in white.
receptor for luteovirus pea enation mosaic virus (PEMV) as alanyl aminopeptidase N (APN) (Linz et al., 2015) used labels on the virus capsid protein (sans RTD) and labels with the RTD domain to determine affinity and location of the receptor. Consistent with the mutants mapped by Byrne et al. (2019), more than 303 coat protein (CP)-label bound to the aphid APN receptor compared to RTD-label. It may be that there are different protein functionalities (CP and CP-RTD) for different receptors as the virus circulates through the aphid. Production of icosahedral VLPs for all the luteovirids investigated was not entirely successful. PEMV capsid protein was expressed in the same manner as BYDV and PLRV, but the particles formed were pleomorphic, lacking icosahedral symmetry. Well-controlled homol-
1736 Structure 27, December 3, 2019
ogy modeling, based on the closely similar sequences of PEMV to BYDV and PLRV, was performed to generate reliable tertiary and quaternary structures for this virus capsid, completing the structures for the three viruses. The use of VLPs for structural models of authentic viruses is now well established and their usefulness is exceptionally important, especially in cases like the luteovirids where sufficient quantities of material are not available for biophysical studies. However, it must be noted that while they are excellent models for structure, the authentic virus particles can have properties different from the VLPs that are important for their biology. A case in point is flock house virus where VLPs crystallized isomorphous with authentic virus and the crystal structures had root mean square deviations of well below an
Byrne, M.J., Steele, J.F.C., Hesketh, E.L., Walden, M., Thompson, R.F., Lomonossoff, G.P., and Ranson, N.A. (2019). Combining Transient Expression and Cryo-EM to Obtain HighResolution Structures of Luteovirid Particles. Structure 27, this issue, 1761–1770. Dreher, T.W., and Miller, W.A. (2006). Translational control in positive strand RNA plant viruses. Virology 344, 185–197. Ho, P.T., Montiel-Garcia, D.J., Wong, J.J., CarrilloTripp, M., Brooks, C.L., 3rd, Johnson, J.E., and Reddy, V.S. (2018). VIPERdb: A Tool for Virus Research. Annu. Rev. Virol. 5, 477–488. Kaplan, I.B., Lee, L., Ripoll, D.R., Palukaitis, P., Gildow, F., and Gray, S.M. (2007). Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission. J. Gen. Virol. 88, 1821–1830. Linz, L.B., Liu, S., Chougule, N.P., and Bonning, B.C. (2015). In Vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector. J. Virol. 89, 11203–11212. Murray, A.A., Sheen, M.R., Veliz, F.A., Fiering, S.N., and Steinmetz, N.F. (2019). In Situ Vaccination of Tumors Using Plant Viral Nanoparticles. Methods Mol. Biol. 2000, 111–124. Peyret, H., and Lomonossoff, G.P. (2013). The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant Mol. Biol. 83, 51–58. Steinmetz, N.F., Lin, T., Lomonossoff, G.P., and Johnson, J.E. (2009). Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. Curr. Top. Microbiol. Immunol. 327, 23–58.