- Email: [email protected]
Current opinion in virology: structural virology
Available online at www.sciencedirect.com
Current opinion in virology: structural virology Editorial overview Mavis Agbandje-McKenna and Richard Kuhn Current Opinion in Virology 2011, 1:81–83 Available online 19th July 2011 1879-6257/$ – see front matter # 2011 Elsevier B.V. All rights reserved. DOI 10.1016/j.coviro.2011.07.001
Mavis Agbandje-McKenna Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA e-mail: [email protected]
Mavis Agbandje-McKenna is professor in the Department of Biochemistry and Molecular Biology at the University of Florida. She is also the director of the Center for Structural Biology within the College of Medicine. Her laboratory uses structural biology and biophysical tools combined with biochemistry and molecular biology to study single-stranded DNA viruses. The goal is to obtain information applicable toward the fundamental understanding of tissue tropism, pathogenicity determination and capsid-mediated host adaptation in addition to the development of disease treatment strategies in the form of virus capsid assembly disruptors and therapeutic gene delivery vectors.
Richard Kuhn Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA
Richard Kuhn is professor and head of the Department of Biological Sciences at Purdue University. He serves as the Gerald and Edna Mann Director of the Bindley Bioscience Center in Purdue’s Discovery Park. His laboratory is interested in envelope virus replication, focusing on alphaviruses and flaviviruses. He and his collaborators have carried out fundamental studies on the structure and assembly of these viruses that include dengue, West Nile, Sindbis and Ross River. In addition, his laboratory is interrogating the relationship between viral and host proteins and the role of lipids in facilitating RNA replication. Knowledge of these processes has allowed his group to pursue strategies for virus intervention. www.sciencedirect.com
Viruses are the causative agents of plant, animal and human diseases and can deliver corrective genomic material to target cells and tissues for genetic disease treatment. They infect members of the three domains of life and recent advances in molecular biology have led to the unearthing of hundreds of endogenous viral genomic sequences. Significantly, even the simplest of viruses have highly efficient coding mechanisms evolved to maximize the use of a limited number of capsid and non-capsid proteins to facilitate functions that are essential for the viral life cycle, including host recognition and entry for infection and protein–protein as well as protein–nucleic acid interactions for assembly. The viral capsid is also the target of host immune surveillance resulting in the evolution of immune evasion strategies. Structural virology is a field of study employing three-dimensional visualization tools, such as X-ray crystallography, nuclear magnetic resonance spectroscopy and cryo-electron microscopy, combined with biochemical, biophysical, and molecular biology approaches, for functional and mechanistic annotation of the steps required for sucessful host infection at the atomic and molecular level. These approaches have provided information on the structural basis of virus capsid–host receptor interactions; the fusion of virus lipid envelopes with host cell membranes; the capsid dynamics required for cellular trafficking; genomic uncoating; virion assembly; nucleic acid replication, transcription and translation; genomic packaging; and host cellular antibody interactions. Structural virology therefore provides information that is essential for dissecting the role of viral and host proteins in viral infection and viral disease pathoegenesis, and provides crucial platforms for the development of viral capsids as cellular genomic and chemical delivery vehicles. The study of viruses using structural tools also has the potential to advance our understanding of cellular biology. Reviews in this issue discuss the role of structural virology in advancing our understanding of viral interactions during infection as well advances in approaches used to study virus structure, particularly giant double-stranded DNA viruses assembled from numerous proteins. The need to combine structural motifs with sequence information for an improved classification of viruses is also reviewed.
Viral attachment and interactions for fusion The recognition of cell surface receptors by viruses is an essential first step in the infection process. For enveloped viruses, the initial attachment step is followed by dynamic transitions of capsid proteins which facilitate the fusion of viral and cellular membranes. The review from Cupelli and Stehle (this issue) discusses the engagement of different cell surface protein and glycan receptors by adenoviruses which enable host infection, as visualized by high resolution X-ray crystallography. These studies have provided fundamental information on the essential virus protein-receptor interactions and the Current Opinion in Virology 2011, 1:81–83
82 Virus structure and function
structural adaptations in different serotypes which facilitate specific interactions. An elegant discussion of the enveloped virus membrane fusion event is provided by Plemper (this issue) in the review of paramyxovirus, flavivirus, and rhabdovirus membrane fusion mediated by glycoproteins incorporated within the viral envelope. These virus groups represent well-studied examples of the three classes of membrane fusion proteins. In all three cases, structural and biophysical studies have played a key role in deciphering the steps involved in bringing viral and cellular membranes together to promote the merging of their lipid bilayers. This is an energy-dependent process, and viral fusion proteins typically have a thermodynamically stable post-fusion conformation that provides the energy to bend the membranes toward each other to facilitate the formation of the fusion pore. However, an understanding of the details of membrane merger is not yet in hand and will require novel imaging techniques such as cryo-electron tomography coupled with new reagents that block steps in the process.
Giant discoveries and advances in technology shed light on common structural ancestry in virion architecture Recent advances in molecular biology have led to the discoveries of a large number of viruses, including those grouped as nucleocytoplasmic large dsDNA viruses (NCLDV). The NCLDVs more closely resemble cells than prototypical small icosahedral viruses. Their structural organizations are being garnered from cryo-electron microscopy and image reconstruction combined with the X-ray crystallographic structures of their major coat protein in addition to other biophysical approaches such as atomic force microscopy. Xiao and Rossmann (this issue) review the latest data on the structural characterization of the NCLDVs which utilized a double jelly-roll motif in the folding of their major coat protein and includes a discussion of specialized and unique surface features which are likely assembled to perform specific functions. Significantly, this review highlights the utility of applying a multitude of structure biology tools for molecular characterization of these giant viruses. In addition to crystallography, significant advances in cryo-electron microscopy approaches, both in single particle reconstruction and tomography have greatly advanced our efforts to study intact virus particles and to study viruses in vitro. Advances in single particle cryoelectron microscopy and image reconstruction have resulted in the determination of intact virus particle structures to near atomic resolution as reviewed by Hryc, Chen, and Chiu (this issue). An example is the structure of Adenovirus for which X-ray crystallography and cryoEM were used to obtain comparable structural resolution. These studies provide important information on the juxtaposition of viral proteins that assemble these macromolecular machines to enable functional annotation, Current Opinion in Virology 2011, 1:81–83
particularly in terms of the capsid assembly and the transition dynamics associated with virion maturation. A novel way to consider virus classification is provided by Krupovic and Bamford (this issue). They discuss structures of the major capsid proteins from double-stranded DNA viruses that infect hosts representing the different domains of life. Although there is diversity in the numbers of virus families and their hosts, they appear to converge on a much more limited set of structural motifs. They therefore, suggest that virus diversity is not so broad if one considers the virion architecture and the capsid protein building blocks that assemble into a virus particle. Furthermore, they suggest that virus classification would be greatly enhanced by using not only sequence but also structure information not only to provide better order but also to reveal evolutionary relationships between viruses.
Electron tomography reveals details of the virus life cycle within the host cell Structural biologists have long sought to examine dynamic viral processes rather than simply relatively static structures of virus particles and their intermediates. Fu and Johnson (this issue) discuss the use of electron tomography to visualize viral processes within the cell from a variety of different viral systems and provide examples of how this relatively new technology is revealing dynamic steps in the process of virus infection, genome replication, and virus assembly and egress. Importantly, these molecular resolution approaches can be coupled to other imaging techniques not only to see the processes but also to identify the viral and host components that participate.
Mechanisms of viral DNA packaging can be revealed by single-molecule approaches Genome packaging following capsid assembly represents an essential step in the life cycle of all viruses and for the dsDNA viruses this process requires the action of specialized groups of motor proteins/nucleic acids which assemble a packaging complex at a special vertex and ATP. Advances in the use of single molecule studies using optical tweezers and fluorescence methods to monitor the packaging process of single DNA molecules into preformed procapsids for the dsDNA bacteriophages have provided significant insight into this process which is likely to have analogies in mammalian dsDNA viruses such as adenovirus. D. Smith reviews (this issue) the advances which have been made toward elucidating the mechanism of dsDNA genome packaging with studies of three bacteriophages, Phi29, Lambda and bacteriophage T4 which have advanced our understanding of this highly coordinated process. The assembly of the herpesvirus capsid provides insight into a complex assembly process and reveals similarities with the assembly processes of dsDNA bacteriophages www.sciencedirect.com
Editorial overview Agbandje-McKenna and Kuhn 83
such as P22 and HK97. In this issue, Jay Brown discusses the contributions of X-ray crystallography and cryo-electron microscopy to elaborate the process of capsid maturation. Although much progress has been made using this system, fundamental questions remain about the association of the herpesvirus capsid with the outer tegument layer and with the process of DNA packaging. It is likely that the single-molecule approaches such as those described by Smith will provide new insights. Furthermore, the use of electron tomography to visualize pleiomorphic structures that are the intermediates in assembly will allow investigators to interrogate this system in finer detail.
Probing further into the lives of viruses After a virus has been assembled and is released from the infected cell, its life cycle is not complete until it finds and enters a new naı¨ve cell. In an animal host, the virus is likely to encounter the defense systems of the host. One of the best studied of these systems is the humoral response that will produce antibodies in an attempt to neutralize virus infection. T. Smith
www.sciencedirect.com
(this issue) describes several examples where a combination of X-ray crystallography and cryo-electron microscopy has been brought to bear on the mechanisms by which antibodies bind to virions and prevent their infection of new cells. In contrast to suggestions from earlier studies, these structural investigations have shown that antibodies do not need to induce conformational changes in their virus target to affect neutralization and that the context of antigen presentation is key for an effective response. Over the last two decades structural biology has contributed in fundamental ways toward our understanding of virus biology. The tools available for structural biology, including new and more advanced instruments and faster computational power, continue to advance the field. As was mentioned above, a static image of a virus particle is no longer sufficient: the field demands the movie version, the High Definition movie version! The next few years will reveal new insights into the molecular dance between pathogen and host and the reality of understanding structure and function will soon be here.
Current Opinion in Virology 2011, 1:81–83
Current opinion in virology: structural virology Editorial overview Mavis Agbandje-McKenna and Richard Kuhn Current Opinion in Virology 2011, 1:81–83 Available online 19th July 2011 1879-6257/$ – see front matter # 2011 Elsevier B.V. All rights reserved. DOI 10.1016/j.coviro.2011.07.001
Mavis Agbandje-McKenna Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA e-mail: [email protected]
Mavis Agbandje-McKenna is professor in the Department of Biochemistry and Molecular Biology at the University of Florida. She is also the director of the Center for Structural Biology within the College of Medicine. Her laboratory uses structural biology and biophysical tools combined with biochemistry and molecular biology to study single-stranded DNA viruses. The goal is to obtain information applicable toward the fundamental understanding of tissue tropism, pathogenicity determination and capsid-mediated host adaptation in addition to the development of disease treatment strategies in the form of virus capsid assembly disruptors and therapeutic gene delivery vectors.
Richard Kuhn Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA
Richard Kuhn is professor and head of the Department of Biological Sciences at Purdue University. He serves as the Gerald and Edna Mann Director of the Bindley Bioscience Center in Purdue’s Discovery Park. His laboratory is interested in envelope virus replication, focusing on alphaviruses and flaviviruses. He and his collaborators have carried out fundamental studies on the structure and assembly of these viruses that include dengue, West Nile, Sindbis and Ross River. In addition, his laboratory is interrogating the relationship between viral and host proteins and the role of lipids in facilitating RNA replication. Knowledge of these processes has allowed his group to pursue strategies for virus intervention. www.sciencedirect.com
Viruses are the causative agents of plant, animal and human diseases and can deliver corrective genomic material to target cells and tissues for genetic disease treatment. They infect members of the three domains of life and recent advances in molecular biology have led to the unearthing of hundreds of endogenous viral genomic sequences. Significantly, even the simplest of viruses have highly efficient coding mechanisms evolved to maximize the use of a limited number of capsid and non-capsid proteins to facilitate functions that are essential for the viral life cycle, including host recognition and entry for infection and protein–protein as well as protein–nucleic acid interactions for assembly. The viral capsid is also the target of host immune surveillance resulting in the evolution of immune evasion strategies. Structural virology is a field of study employing three-dimensional visualization tools, such as X-ray crystallography, nuclear magnetic resonance spectroscopy and cryo-electron microscopy, combined with biochemical, biophysical, and molecular biology approaches, for functional and mechanistic annotation of the steps required for sucessful host infection at the atomic and molecular level. These approaches have provided information on the structural basis of virus capsid–host receptor interactions; the fusion of virus lipid envelopes with host cell membranes; the capsid dynamics required for cellular trafficking; genomic uncoating; virion assembly; nucleic acid replication, transcription and translation; genomic packaging; and host cellular antibody interactions. Structural virology therefore provides information that is essential for dissecting the role of viral and host proteins in viral infection and viral disease pathoegenesis, and provides crucial platforms for the development of viral capsids as cellular genomic and chemical delivery vehicles. The study of viruses using structural tools also has the potential to advance our understanding of cellular biology. Reviews in this issue discuss the role of structural virology in advancing our understanding of viral interactions during infection as well advances in approaches used to study virus structure, particularly giant double-stranded DNA viruses assembled from numerous proteins. The need to combine structural motifs with sequence information for an improved classification of viruses is also reviewed.
Viral attachment and interactions for fusion The recognition of cell surface receptors by viruses is an essential first step in the infection process. For enveloped viruses, the initial attachment step is followed by dynamic transitions of capsid proteins which facilitate the fusion of viral and cellular membranes. The review from Cupelli and Stehle (this issue) discusses the engagement of different cell surface protein and glycan receptors by adenoviruses which enable host infection, as visualized by high resolution X-ray crystallography. These studies have provided fundamental information on the essential virus protein-receptor interactions and the Current Opinion in Virology 2011, 1:81–83
82 Virus structure and function
structural adaptations in different serotypes which facilitate specific interactions. An elegant discussion of the enveloped virus membrane fusion event is provided by Plemper (this issue) in the review of paramyxovirus, flavivirus, and rhabdovirus membrane fusion mediated by glycoproteins incorporated within the viral envelope. These virus groups represent well-studied examples of the three classes of membrane fusion proteins. In all three cases, structural and biophysical studies have played a key role in deciphering the steps involved in bringing viral and cellular membranes together to promote the merging of their lipid bilayers. This is an energy-dependent process, and viral fusion proteins typically have a thermodynamically stable post-fusion conformation that provides the energy to bend the membranes toward each other to facilitate the formation of the fusion pore. However, an understanding of the details of membrane merger is not yet in hand and will require novel imaging techniques such as cryo-electron tomography coupled with new reagents that block steps in the process.
Giant discoveries and advances in technology shed light on common structural ancestry in virion architecture Recent advances in molecular biology have led to the discoveries of a large number of viruses, including those grouped as nucleocytoplasmic large dsDNA viruses (NCLDV). The NCLDVs more closely resemble cells than prototypical small icosahedral viruses. Their structural organizations are being garnered from cryo-electron microscopy and image reconstruction combined with the X-ray crystallographic structures of their major coat protein in addition to other biophysical approaches such as atomic force microscopy. Xiao and Rossmann (this issue) review the latest data on the structural characterization of the NCLDVs which utilized a double jelly-roll motif in the folding of their major coat protein and includes a discussion of specialized and unique surface features which are likely assembled to perform specific functions. Significantly, this review highlights the utility of applying a multitude of structure biology tools for molecular characterization of these giant viruses. In addition to crystallography, significant advances in cryo-electron microscopy approaches, both in single particle reconstruction and tomography have greatly advanced our efforts to study intact virus particles and to study viruses in vitro. Advances in single particle cryoelectron microscopy and image reconstruction have resulted in the determination of intact virus particle structures to near atomic resolution as reviewed by Hryc, Chen, and Chiu (this issue). An example is the structure of Adenovirus for which X-ray crystallography and cryoEM were used to obtain comparable structural resolution. These studies provide important information on the juxtaposition of viral proteins that assemble these macromolecular machines to enable functional annotation, Current Opinion in Virology 2011, 1:81–83
particularly in terms of the capsid assembly and the transition dynamics associated with virion maturation. A novel way to consider virus classification is provided by Krupovic and Bamford (this issue). They discuss structures of the major capsid proteins from double-stranded DNA viruses that infect hosts representing the different domains of life. Although there is diversity in the numbers of virus families and their hosts, they appear to converge on a much more limited set of structural motifs. They therefore, suggest that virus diversity is not so broad if one considers the virion architecture and the capsid protein building blocks that assemble into a virus particle. Furthermore, they suggest that virus classification would be greatly enhanced by using not only sequence but also structure information not only to provide better order but also to reveal evolutionary relationships between viruses.
Electron tomography reveals details of the virus life cycle within the host cell Structural biologists have long sought to examine dynamic viral processes rather than simply relatively static structures of virus particles and their intermediates. Fu and Johnson (this issue) discuss the use of electron tomography to visualize viral processes within the cell from a variety of different viral systems and provide examples of how this relatively new technology is revealing dynamic steps in the process of virus infection, genome replication, and virus assembly and egress. Importantly, these molecular resolution approaches can be coupled to other imaging techniques not only to see the processes but also to identify the viral and host components that participate.
Mechanisms of viral DNA packaging can be revealed by single-molecule approaches Genome packaging following capsid assembly represents an essential step in the life cycle of all viruses and for the dsDNA viruses this process requires the action of specialized groups of motor proteins/nucleic acids which assemble a packaging complex at a special vertex and ATP. Advances in the use of single molecule studies using optical tweezers and fluorescence methods to monitor the packaging process of single DNA molecules into preformed procapsids for the dsDNA bacteriophages have provided significant insight into this process which is likely to have analogies in mammalian dsDNA viruses such as adenovirus. D. Smith reviews (this issue) the advances which have been made toward elucidating the mechanism of dsDNA genome packaging with studies of three bacteriophages, Phi29, Lambda and bacteriophage T4 which have advanced our understanding of this highly coordinated process. The assembly of the herpesvirus capsid provides insight into a complex assembly process and reveals similarities with the assembly processes of dsDNA bacteriophages www.sciencedirect.com
Editorial overview Agbandje-McKenna and Kuhn 83
such as P22 and HK97. In this issue, Jay Brown discusses the contributions of X-ray crystallography and cryo-electron microscopy to elaborate the process of capsid maturation. Although much progress has been made using this system, fundamental questions remain about the association of the herpesvirus capsid with the outer tegument layer and with the process of DNA packaging. It is likely that the single-molecule approaches such as those described by Smith will provide new insights. Furthermore, the use of electron tomography to visualize pleiomorphic structures that are the intermediates in assembly will allow investigators to interrogate this system in finer detail.
Probing further into the lives of viruses After a virus has been assembled and is released from the infected cell, its life cycle is not complete until it finds and enters a new naı¨ve cell. In an animal host, the virus is likely to encounter the defense systems of the host. One of the best studied of these systems is the humoral response that will produce antibodies in an attempt to neutralize virus infection. T. Smith
www.sciencedirect.com
(this issue) describes several examples where a combination of X-ray crystallography and cryo-electron microscopy has been brought to bear on the mechanisms by which antibodies bind to virions and prevent their infection of new cells. In contrast to suggestions from earlier studies, these structural investigations have shown that antibodies do not need to induce conformational changes in their virus target to affect neutralization and that the context of antigen presentation is key for an effective response. Over the last two decades structural biology has contributed in fundamental ways toward our understanding of virus biology. The tools available for structural biology, including new and more advanced instruments and faster computational power, continue to advance the field. As was mentioned above, a static image of a virus particle is no longer sufficient: the field demands the movie version, the High Definition movie version! The next few years will reveal new insights into the molecular dance between pathogen and host and the reality of understanding structure and function will soon be here.
Current Opinion in Virology 2011, 1:81–83