RNA Packaging in the Cystovirus Bacteriophages: Dynamic Interactions during Capsid Maturation.

The bacteriophage family Cystoviridae consists of a single genus, Cystovirus, that is lipid-containing with three double-stranded RNA (ds-RNA) genome segments. With regard to the segmented dsRNA genome, they resemble the family Reoviridae. Therefore, the Cystoviruses have long served as a simple model for reovirus assembly. This review focuses on important developments in the study of the RNA packaging and replication mechanisms, emphasizing the structural conformations and dynamic changes during maturation of the five proteins required for viral RNA synthesis, P1, P2, P4, P7, and P8. Together these proteins constitute the procapsid/polymerase complex (PC) and nucleocapsid (NC) of the Cystoviruses. During viral assembly and RNA packaging, the five proteins must function in a coordinated fashion as the PC and NC undergo expansion with significant position translation. The review emphasizes this facet of the viral assembly process and speculates on areas suggestive of additional research efforts.

Cryo-Electron Microscopy Structure of Seneca Valley Virus Procapsid.

Seneca Valley virus (SVV), like some other members of the Picornaviridae, forms naturally occurring empty capsids, known as procapsids. Procapsids have the same antigenicity as full virions, so they present an interesting possibility for the formation of stable virus-like particles. Interestingly, although SVV is a livestock pathogen, it has also been found to preferentially infect tumor cells and is being explored for use as a therapeutic agent in the treatment of small-cell lung cancers. Here we used cryo-electron microscopy to investigate the procapsid structure and describe the transition of capsid protein VP0 to the cleaved forms of VP4 and VP2. We show that the SVV receptor binds the procapsid, as evidence of its native antigenicity. In comparing the procapsid structure to that of the full virion, we also show that a cage of RNA serves to stabilize the inside surface of the virus, thereby making it more acid stable.IMPORTANCE Viruses are extensively studied to help us understand infection and disease. One of the by-products of some virus infections are the naturally occurring empty virus capsids (containing no genome), termed procapsids, whose function remains unclear. Here we investigate the structure and formation of the procapsids of Seneca Valley virus, to better understand how they form, what causes them to form, how they behave, and how we can make use of them. One potential benefit of this work is the modification of the procapsid to develop it for targeted in vivo delivery of therapeutics or to make a stable vaccine against SVV, which could be of great interest to the agricultural industry.

The Apical Region of the Herpes Simplex Virus Major Capsid Protein Promotes Capsid Maturation.

The herpesvirus capsid assembles in the nucleus as an immature procapsid precursor built around viral scaffold proteins. The event that initiates procapsid maturation is unknown, but it is dependent upon activation of the VP24 internal protease. Scaffold cleavage triggers angularization of the shell and its decoration with the VP26 and pUL25 capsid-surface proteins. In both the procapsid and mature angularized capsid, the apical region of the major capsid protein (VP5) is surface exposed. We investigated whether the VP5 apical region contributes to intracellular transport dynamics following entry into primary sensory neurons and also tested the hypothesis that conserved negatively charged amino acids in the apical region contribute to VP26 acquisition. To our surprise, neither hypothesis proved true. Instead, mutation of glutamic acid residues in the apical region delayed viral propagation and induced focal capsid accumulations in nuclei. Examination of capsid morphogenesis based on epitope unmasking, capsid composition, and ultrastructural analysis indicated that these clusters consisted of procapsids. The results demonstrate that, in addition to established events that occur inside the capsid, the exterior capsid shell promotes capsid morphogenesis and maturation.IMPORTANCE Herpesviruses assemble capsids and encapsidate their genomes by a process that is unlike those of other mammalian viruses but is similar to those of some bacteriophage. Many important aspects of herpesvirus morphogenesis remain enigmatic, including how the capsid shell matures into a stable angularized configuration. Capsid maturation is triggered by activation of a protease that cleaves an internal protein scaffold. We report on the fortuitous discovery that a region of the major capsid protein that is exposed on the outer surface of the capsid also contributes to capsid maturation, demonstrating that the morphogenesis of the capsid shell from its procapsid precursor to the mature angularized form is dependent upon internal and external components of the megastructure.

Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release.

The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. IMPORTANCE: Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV.

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain.

The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the ß-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1-2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo By contrast, the mutants have only minor effects on capsid expansion or stability in vitro The effects of the Asp-302-His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits.

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions.

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.

Binding of Coxsackievirus A21 procapsids to immobilized glutathione depends on cell culture conditions during infection.

A prototype strain of Coxsackievirus A21 (CVA21) is under clinical evaluation as an oncolytic virus immunotherapy. To improve scalability of the manufacturing process, an affinity chromatography purification method was developed using immobilized glutathione resin that captured infectious CVA21 virions from cell culture harvests with high recovery and impurity clearance. Unexpectedly, the binding of empty CVA21 procapsids depended on production cell culture conditions during infection including temperature, presence of serum in the media, and production cell line. At 37 °C and 2% serum during infection, procapsids flowed-through while infectious virions bound and were recovered at >95% yield in the chromatography elution. However, at sub-physiological temperature or after removal of serum at infection, both procapsids and mature virions bound and co-eluted from the immobilized glutathione ligand. This work may improve the understanding of CVA21 capsid assembly and presents an efficient purification method that may be applied to picornaviruses that interact with intracellular GSH.

Structural basis of bacteriophage lambda capsid maturation.

Bacteriophage lambda is an excellent model system for studying capsid assembly of double-stranded DNA (dsDNA) bacteriophages, some dsDNA archaeal viruses, and herpesviruses. HK97 fold coat proteins initially assemble into a precursor capsid (procapsid) and subsequent genome packaging triggers morphological expansion of the shell. An auxiliary protein is required to stabilize the expanded capsid structure. To investigate the capsid maturation mechanism, we determined the cryo-electron microscopy structures of the bacteriophage lambda procapsid and mature capsid at 3.88 Å and 3.76 Å resolution, respectively. Besides primarily rigid body movements of common features of the major capsid protein gpE, large-scale structural rearrangements of other domains occur simultaneously. Assembly of intercapsomers within the procapsid is facilitated by layer-stacking effects at 3-fold vertices. Upon conformational expansion of the capsid shell, the missing top layer is fulfilled by cementing the gpD protein against the internal pressure of DNA packaging. Our structures illuminate the assembly mechanisms of dsDNA viruses.

Kaposi's sarcoma-associated herpesvirus ORF17 plays a key role in capsid maturation.

Kaposi's sarcoma-associated herpesvirus is a human rhadinovirus of the gammaherpesvirus sub-family. Although herpesviruses are well-studied models of capsid formation and its processes, those of KSHV remain unknown. KSHV ORF17 encoding the viral protease precursor (ORF17-prePR) is thought to contribute to capsid formation; however, functional information is largely unknown. Here, we evaluated the role of ORF17 during capsid formation by generating ORF17-deficient and ORF17 protease-dead KSHV. Both mutants showed a decrease in viral production but not DNA replication. ORF17 R-mut, with a point-mutation at the restriction or release site (R-site) by which ORF17-prePR can be functionally cleaved into a protease (ORF17-PR) and an assembly region (ORF17-pAP/-AP), failed to play a role in viral production. Furthermore, wild type KSHV produced a mature capsid, whereas ORF17-deficient and protease-dead KSHV produced a B-capsid, (i.e., a closed body possessing a circular inner structure). Therefore, ORF17 and its protease function are essential for appropriate capsid maturation.

Assembly of bacteriophage P2 capsids from capsid protein fused to internal scaffolding protein.

Most tailed bacteriophages with double-stranded DNA genomes code for a scaffolding protein, which is required for capsid assembly, but is removed during capsid maturation and DNA packaging. The gpO scaffolding protein of bacteriophage P2 also doubles as a maturation protease, while the scaffolding activity is confined to a 90 residue C-terminal "scaffolding" domain. Bacteriophage HK97 lacks a separate scaffolding protein; instead, an N-terminal "delta" domain in the capsid protein appears to serve an analogous role. We asked whether the C-terminal scaffolding domain of gpO could work as a delta domain when fused to the gpN capsid protein. Varying lengths of C-terminal sequences from gpO were fused to the N-terminus of gpN and expressed in E. coli. The presence of just the 41 C-terminal residues of gpO increased the fidelity of assembly and promoted the formation of closed shells, but the shells formed were predominantly small, 40 nm shells, compared to the normal, 55 nm P2 procapsid shells. Larger scaffolding domains fused to gpN caused the formation of shells of varying size and shape. The results suggest that while fusing the scaffolding protein to the capsid protein assists in shell closure, it also restricts the conformational variability of the capsid protein.

NMR assignments for monomeric phage L decoration protein.

Phage L encodes a trimeric 43 kDa decoration protein (Dec) that noncovalently binds and stabilizes the capsids of the homologous phages L and P22 in vitro. At physiological pH Dec was unsuitable for NMR. We were able to obtain samples amenable for NMR spectroscopy by unfolding Dec to pH 2 and refolding it to pH 4. Our unfolding/refolding protocol converted trimeric Dec to a folded 14.4 kDa monomer. We verified that the acid-unfolding protocol did not perturb the secondary structure, or the capsid-binding function of refolded Dec. We were able to obtain complete 1H, 15N, and 13C assignments for the Dec monomer, as well as information on its secondary structure and dynamics based on chemical shift assignments. The assigned NMR spectrum is being used to determine the three-dimensional structure of Dec, which is important for understanding how the trimer binds phage capsids and for the use of the protein as a platform for phage-display nanotechnology.

Acquiring Structural Information on Virus Particles with Charge Detection Mass Spectrometry.

Charge detection mass spectrometry (CDMS) is a single-molecule technique particularly well-suited to measuring the mass and charge distributions of heterogeneous, MDa-sized ions. In this work, CDMS has been used to analyze the assembly products of two coat protein variants of bacteriophage P22. The assembly products show broad mass distributions extending from 5 to 15 MDa for A285Y and 5 to 25 MDa for A285T coat protein variants. Because the charge of large ions generated by electrospray ionization depends on their size, the charge can be used to distinguish hollow shells from more compact structures. A285T was found to form T = 4 and T = 7 procapsids, and A285Y makes a small number of T = 3 and T = 4 procapsids. Owing to the decreased stability of the A285Y and A285T particles, chemical cross-linking was required to stabilize them for electrospray CDMS.Graphical Abstract.

Common Evolutionary Origin of Procapsid Proteases, Phage Tail Tubes, and Tubes of Bacterial Type VI Secretion Systems.

Many large viruses, including tailed dsDNA bacteriophages and herpesviruses, assemble their capsids via formation of precursors, called procapsids or proheads. The prohead has an internal core, made of scaffolding proteins, and an outer shell, formed by the major capsid protein. The prohead usually contains a protease, which is activated during capsid maturation to destroy the inner core and liberate space for the genome. Here, we report a 2.0 Å resolution structure of the pentameric procapsid protease of bacteriophage T4, gene product (gp)21. The structure corresponds to the enzyme's pre-active state in which its N-terminal region blocks the catalytic center, demonstrating that the activation mechanism involves self-cleavage of nine N-terminal residues. We describe similarities and differences between T4 gp21 and related herpesvirus proteases. We found that gp21 and the herpesvirus proteases have similarity with proteins forming the tubes of phage tails and bacterial type VI secretion systems, suggesting their common evolutionary origin.

NMR assignments for the insertion domain of bacteriophage CUS-3 coat protein.

CUS-3 is a P22-like tailed dsDNA bacteriophage that infects Escherichia coli serotype K1. The CUS-3 coat protein, which forms the icosahedral capsid, has a conserved HK97-fold but with a non-conserved accessory domain known as the insertion domain (I-domain). Sequence alignment of the coat proteins from CUS-3 and P22 shows higher sequence similarity for the I-domains (35 %) than for the HK97-cores, suggesting the I-domains play important functional roles. The I-domain of the P22 coat protein, which has an NMR structure comprised of a six-stranded ß-barrel, has been shown to govern the assembly, stability and size of the resulting capsid particles. Here, we report the (1)H, (15)N, and (13)C assignments for the I-domain from the coat protein of bacteriophage CUS-3. The secondary structure and dynamics of the CUS-3 I-domain, predicted from the assigned NMR chemical shifts, agree with those of the P22 I-domain, suggesting the CUS-3 and P22 I-domains may have similar structures and functions in capsid assembly.

ϕ29 Scaffolding and connector structure-function relationship studied by trans-complementation.

A dodecamer of connector protein forms a conduit at a unique five-fold vertex in the capsid of many dsDNA-containing viruses providing the means for DNA entry and egress. The molecular mechanism guiding the incorporation of one connector per procapsid remains obscure; however, a recent bacteriophage ϕ29 model suggests that incorporation is coupled to nucleation between the connector and scaffolding proteins and particular amino acids may promote interactions between the two proteins. To test this model in vivo, a trans-complementation system using cloned scaffolding genes was implemented and tested for the ability to complement a ϕ29 amber-scaffolding strain. Wild type scaffolding gene induction resulted in efficient virion production, whereas synthesis of mutant scaffolding proteins displayed various phenotypes. Biochemical analyses of the resultant particles substantiate the previously identified amino acid residues in connector incorporation. Furthermore, kinetic studies of virion production using the in vivo trans-complementation system support the nucleation model.