Principles for enhancing virus capsid capacity and stability from a thermophilic virus capsid structure

Abstract The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-Å resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T = 7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased with a larger, flatter major capsid protein. Given these results, we predict decreased icosahedral complexity (i.e. T ≤ 7) leads to a more stable capsid assembly.

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Principles for enhancing virus capsid capacity and stability from a thermophilic virus capsid structure

10.1101/473264 ◽

2018 ◽

Cited By ~ 2

Author(s):

Nicholas P. Stone ◽

Gabriel Demo ◽

Emily Agnello ◽

Brian A. Kelch

Keyword(s):

Major Capsid Protein ◽

Extreme Environment ◽

Structural Basis ◽

Genome Packaging ◽

Virus Capsid ◽

Dna Viruses ◽

Double Stranded Dna ◽

High Internal Pressure ◽

Extracellular Environment ◽

Catch Bonds

SUMMARYThe capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we used cryoelectron microscopy to determine the capsid structure of the thermostable phage P74-26 to 2.8-Å resolution. We find the P74-26 capsid exhibits an overall architecture that is very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals ‘lasso’-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T=7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased through a novel mechanism with a larger, flatter major capsid protein. Our results suggest that decreased icosahedral complexity (i.e. lower T number) leads to a more stable capsid assembly.

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Adintoviruses: A Proposed Animal-Tropic Family of Midsize Eukaryotic Linear dsDNA (MELD) Viruses

Virus Evolution ◽

10.1093/ve/veaa055 ◽

2020 ◽

Cited By ~ 1

Author(s):

Gabriel J Starrett ◽

Michael J Tisza ◽

Nicole L Welch ◽

Anna K Belford ◽

Alberto Peretti ◽

...

Keyword(s):

Genome Packaging ◽

Metagenomic Sequence ◽

Dna Viruses ◽

Diverse Range ◽

Integrase Gene ◽

Data Mining Approach ◽

Double Stranded Dna ◽

Wide Range ◽

Linear Dsdna ◽

Dsdna Viruses

Abstract Polintons (also known as Mavericks) were initially identified as a widespread class of eukaryotic transposons named for their hallmark type B DNA polymerase and retrovirus-like integrase genes. It has since been recognized that many polintons encode possible capsid proteins and viral genome-packaging ATPases similar to those of a diverse range of double-stranded DNA (dsDNA) viruses. This supports the inference that at least some polintons are actually viruses capable of cell-to-cell spread. At present, there are no polinton-associated capsid protein genes annotated in public sequence databases. To rectify this deficiency, we used a data-mining approach to investigate the distribution and gene content of polinton-like elements and related DNA viruses in animal genomic and metagenomic sequence datasets. The results define a discrete family-like clade of viruses with two genus-level divisions. We propose the family name Adintoviridae, connoting similarities to adenovirus virion proteins and the presence of a retrovirus-like integrase gene. Although adintovirus-class PolB sequences were detected in datasets for fungi and various unicellular eukaryotes, sequences resembling adintovirus virion proteins and accessory genes appear to be restricted to animals. Degraded adintovirus sequences are endogenized into the germlines of a wide range of animals, including humans.

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NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

10.1101/2020.07.06.188573 ◽

2020 ◽

Cited By ~ 2

Author(s):

Bryon P. Mahler ◽

Pawel J. Bujalowski ◽

Huzhang Mao ◽

Erik A. Dill ◽

Paul J. Jardine ◽

...

Keyword(s):

Conformational Changes ◽

Molecular Motors ◽

Solution Structure ◽

Nuclease Activity ◽

Structural Basis ◽

Binding Assays ◽

Dna Viruses ◽

Double Stranded Dna ◽

Fluorescence Binding ◽

Insight Into

SummaryDouble-stranded DNA viruses use ATP-powered molecular motors to package their genomes. To do so, these motors must efficiently transition between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes dimerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination.

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ATP serves as a nucleotide switch coupling the genome maturation and packaging motor complexes of a virus assembly machine

Nucleic Acids Research ◽

10.1093/nar/gkaa205 ◽

2020 ◽

Vol 48 (9) ◽

pp. 5006-5015

Author(s):

Qin Yang ◽

Carlos E Catalano

Keyword(s):

Atp Hydrolysis ◽

Virus Assembly ◽

Genome Packaging ◽

Dna Viruses ◽

Motor Complex ◽

Double Stranded Dna ◽

Tightly Coupled ◽

Switch Mechanism ◽

Genome Maturation ◽

Dsdna Viruses

Abstract The assembly of double-stranded DNA viruses, from phages to herpesviruses, is strongly conserved. Terminase enzymes processively excise and package monomeric genomes from a concatemeric DNA substrate. The enzymes cycle between a stable maturation complex that introduces site-specific nicks into the duplex and a dynamic motor complex that rapidly translocates DNA into a procapsid shell, fueled by ATP hydrolysis. These tightly coupled reactions are catalyzed by terminase assembled into two functionally distinct nucleoprotein complexes; the maturation complex and the packaging motor complex, respectively. We describe the effects of nucleotides on the assembly of a catalytically competent maturation complex on viral DNA, their effect on maturation complex stability and their requirement for the transition to active packaging motor complex. ATP plays a major role in regulating all of these activities and may serve as a ‘nucleotide switch’ that mediates transitions between the two complexes during processive genome packaging. These biological processes are recapitulated in all of the dsDNA viruses that package monomeric genomes from concatemeric DNA substrates and the nucleotide switch mechanism may have broad biological implications with respect to virus assembly mechanisms.

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Cryo-EM analysis of a viral portal protein in situ reveals a switch in the DNA tunnel

10.1101/713933 ◽

2019 ◽

Author(s):

Oliver W. Bayfield ◽

Alasdair C. Steven ◽

Alfred A. Antson

Keyword(s):

Surface Properties ◽

Dynamic Processes ◽

Viral Capsid ◽

Capsid Assembly ◽

Genome Packaging ◽

Dna Viruses ◽

Portal Protein ◽

Double Stranded Dna ◽

Protein Functions

The portal protein is a key component of many double-stranded DNA viruses, governing capsid assembly and genome packaging. Twelve subunits of the portal protein form a ring with a central tunnel, through which DNA is translocated into the capsid. It is unknown how the portal protein functions as a gatekeeper, preventing DNA slippage, whilst allowing its passage into the capsid through its central tunnel, and how these processes can be controlled by capsid and motor proteins. A cryo-EM structure of a portal protein, determined in situ for immature capsids of thermostable bacteriophage P23-45, suggests how domain adjustments can be coupled with a switching of properties of the DNA tunnel. Of particular note is an inversion of the conformation of portal loops which define the tunnel’s constriction, accompanied by a switching of surface properties from hydrophobic to hydrophilic. These observations indicate how translocation of DNA into the viral capsid can be modulated by changes in the properties and size of the central tunnel and how the changing pattern of protein–capsid interactions across a symmetry-mismatched interface can facilitate these dynamic processes.

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Cryo-EM structure in situ reveals a molecular switch that safeguards virus against genome loss

eLife ◽

10.7554/elife.55517 ◽

2020 ◽

Vol 9 ◽

Author(s):

Oliver W Bayfield ◽

Alasdair C Steven ◽

Alfred A Antson

Keyword(s):

Protein Interactions ◽

Bound State ◽

Molecular Switch ◽

Protein Protein Interactions ◽

Genome Packaging ◽

Dna Viruses ◽

Portal Protein ◽

Double Stranded Dna ◽

Protein Functions

The portal protein is a key component of many double-stranded DNA viruses, governing capsid assembly and genome packaging. Twelve subunits of the portal protein define a tunnel, through which DNA is translocated into the capsid. It is unknown how the portal protein functions as a gatekeeper, preventing DNA slippage, whilst allowing its passage into the capsid, and how these processes are controlled. A cryo-EM structure of the portal protein of thermostable virus P23-45, determined in situ in its procapsid-bound state, indicates a mechanism that naturally safeguards the virus against genome loss. This occurs via an inversion of the conformation of the loops that define the constriction in the central tunnel, accompanied by a hydrophilic–hydrophobic switch. The structure also shows how translocation of DNA into the capsid could be modulated by a changing mode of protein–protein interactions between portal and capsid, across a symmetry-mismatched interface.

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NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

Nucleic Acids Research ◽

10.1093/nar/gkaa874 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11737-11749 ◽

Cited By ~ 1

Author(s):

Bryon P Mahler ◽

Paul J Bujalowski ◽

Huzhang Mao ◽

Erik A Dill ◽

Paul J Jardine ◽

...

Keyword(s):

Conformational Changes ◽

Molecular Motors ◽

Solution Structure ◽

Nuclease Activity ◽

Structural Basis ◽

Binding Assays ◽

Dna Viruses ◽

Double Stranded Dna ◽

Fluorescence Binding ◽

Insight Into

Abstract Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.

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Structure of faustovirus, a large dsDNA virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1523999113 ◽

2016 ◽

Vol 113 (22) ◽

pp. 6206-6211 ◽

Cited By ~ 47

Author(s):

Thomas Klose ◽

Dorine G. Reteno ◽

Samia Benamar ◽

Adam Hollerbach ◽

Philippe Colson ◽

...

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Dsdna Virus ◽

Dna Virus ◽

Dna Viruses ◽

Double Stranded Dna ◽

Cryo Electron Microscopy ◽

Maturation Stages ◽

Jelly Roll ◽

Protein Shell

Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. Here we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unit of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. This complexity might help the virus to rapidly adapt to new environments or hosts.

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Structural basis for genome packaging, retention, and ejection in human cytomegalovirus

Nature Communications ◽

10.1038/s41467-021-24820-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhihai Li ◽

Jingjing Pang ◽

Lili Dong ◽

Xuekui Yu

Keyword(s):

Human Cytomegalovirus ◽

Conformational Changes ◽

Major Capsid Protein ◽

Coiled Coil ◽

Cell Entry ◽

Structural Basis ◽

Genome Packaging ◽

Human Herpesviruses ◽

Viral Cell Entry

AbstractHow the human cytomegalovirus (HCMV) genome—the largest among human herpesviruses—is packaged, retained, and ejected remains unclear. We present the in situ structures of the symmetry-mismatched portal and the capsid vertex-specific components (CVSCs) of HCMV. The 5-fold symmetric 10-helix anchor—uncommon among known portals—contacts the portal-encircling DNA, which is presumed to squeeze the portal as the genome packaging proceeds. We surmise that the 10-helix anchor dampens this action to delay the portal reaching a “head-full” packaging state, thus facilitating the large genome to be packaged. The 6-fold symmetric turret, latched via a coiled coil to a helix from a major capsid protein, supports the portal to retain the packaged genome. CVSCs at the penton vertices—presumed to increase inner capsid pressure—display a low stoichiometry, which would aid genome retention. We also demonstrate that the portal and capsid undergo conformational changes to facilitate genome ejection after viral cell entry.

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Adintoviruses: An Animal-Tropic Family of Midsize Eukaryotic Linear dsDNA (MELD) Viruses

10.1101/697771 ◽

2019 ◽

Author(s):

Gabriel J. Starrett ◽

Michael J. Tisza ◽

Nicole L. Welch ◽

Anna K. Belford ◽

Alberto Peretti ◽

...

Keyword(s):

Genome Packaging ◽

Metagenomic Sequence ◽

Dna Viruses ◽

Diverse Range ◽

Data Mining Approach ◽

Double Stranded Dna ◽

Wide Range ◽

The Family ◽

Linear Dsdna ◽

Dsdna Viruses

AbstractPolintons (also known as Mavericks) were initially identified as a widespread class of eukaryotic transposons named for their hallmark type B DNA polymerase and retrovirus-like integrase genes. It has since been recognized that many polintons encode possible capsid proteins and viral genome-packaging ATPases similar to those of a diverse range of double-stranded DNA (dsDNA) viruses. This supports the inference that at least some polintons are viruses that remain capable of cell-to-cell spread. At present, there are no polinton-associated capsid protein genes annotated in public sequence databases. To rectify this deficiency, we used a data-mining approach to investigate the distribution and gene content of polinton-like elements and related DNA viruses in animal genomic and metagenomic sequence datasets. The results define a discrete family-like clade of animal-specific viruses with two genus-level divisions. We suggest the family name Adintoviridae, connoting similarities to adenovirus virion proteins and the presence of a retrovirus-like integrase gene. Although adintovirus-class PolB sequences were detected in datasets for fungi and various unicellular eukaryotes, sequences resembling adintovirus virion proteins and accessory genes appear to be restricted to animals. Degraded adintovirus sequences are endogenized into the germlines of a wide range of animals, including humans.

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