DNA Binding and Condensation Properties of the Herpes Simplex Virus Type 1 Triplex Protein VP19C

Herpesvirus capsids are regular icosahedrons with a diameter of a 125 nm and are made up of 162 capsomeres arranged on a T = 16 lattice. The capsomeres (VP5) interact with the triplex structure, which is a unique structural feature of herpesvirus capsid shells. The triplex is a heterotrimeric complex; one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent capsomeres. VP19C interacts with VP23 and with the major capsid protein VP5 and is required for the nuclear localization of VP23. Mutation of VP19C results in the abrogation of capsid shell synthesis. Analysis of the sequence of VP19C showed the N-terminus of VP19C is very basic and glycine rich. It was hypothesized that this domain could potentially bind to DNA. In this study an electrophoretic mobility shift assay (EMSA) and a DNA condensation assay were performed to demonstrate that VP19C can bind DNA. Purified VP19C was able to bind to both a DNA fragment of HSV-1 origin as well as a bacterial plasmid sequence indicating that this activity is non-specific. Ultra-structural imaging of the nucleo-protein complexes revealed that VP19C condensed the DNA and forms toroidal DNA structures. Both the DNA binding and condensing properties of VP19C were mapped to the N-terminal 72 amino acids of the protein. Mutational studies revealed that the positively charged arginine residues in this N-terminal domain are required for this binding. This DNA binding activity, which resides in a non-conserved region of the protein could be required for stabilization of HSV-1 DNA association in the capsid shell.     

Structural features of the scaffold interaction domain at the N terminus of the major capsid protein (VP5) of herpes simplex virus type 1

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 capsid. A key interaction occurs between the C terminus of the scaffold protein and the N terminus of the major capsid protein (VP5). Results from alanine-scanning mutagenesis of hydrophobic residues in the N terminus of VP5 revealed seven residues (I27, L35, F39, L58, L65, L67, and L71) that reside in two predicted alpha helices (helix 1(22-42) and helix 2(58-72)) that are important for this bimolecular interaction. The goal of the present study was to further characterize the VP5 scaffold interaction domain (SID). Amino acids at the seven positions were replaced with L, M, V or P (I27); I, M, V, or P (L35, L58, L65, L67, and L71); and H, W, Y, or L (F39). Replacement with a hydrophobic side chain did not affect the interaction with scaffold protein in yeast cells or the ability of a virus specifying the mutation from replicating in cells. The mutation to the proline side chain abolished the interaction in all cases and was lethal for virus replication. Mutant viruses with proline substitutions in helix 1(22-42) at positions 27 and 35 assembled large open capsid shells that did not attain closure. Proline substitutions in helix 2(58-72) at either position 59, 65, or 67 abolished the accumulation of VP5 protein, and, at 58 and 71, although VP5 did accumulate, capsid shells were not assembled. Thus, the second SID, SID2, is highly structured, and this alpha helix (helix 2(58-72)) is likely involved in capsomere-capsomere interactions during shell accretion. Conserved glycine G59 in helix 2(58-72) was also mutated. G59 may act as a flexible "hinge" in helix 2(58-72) because decreasing the movement of this side chain by replacement with valine impaired capsid assembly. Thus, the N terminus of VP5 and the alpha helices embedded in this domain, as in the capsid shell proteins of some double-stranded DNA phages, are a key regulator of shell accretion and stabilization.     

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T = 3 capsid protein leads to larger T = 4 capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.     

Self-association of herpes simplex virus type 1 ICP35 is via coiled-coil interactions and promotes stable interaction with the major capsid protein.

The ordered copolymerization of viral proteins to form the herpes simplex virus (HSV) capsid occurs within the nucleus of the infected cell and is a complex process involving the products of at least six viral genes. In common with capsid assembly in double-stranded DNA bacteriophages, HSV capsid assembly proceeds via the assembly of an outer capsid shell around an interior scaffold. This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA. The interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processing of these proteins is essential for and accompanies capsid maturation. The assembly protein (ICP35) is the primary component of the scaffold, and previous studies have demonstrated it to be capable of intermolecular association with itself and with the major capsid protein, VP5. We have defined structural elements within ICP35 which are responsible for intermolecular self-association and for interaction with VP5. Yeast (Saccharomyces cerevisiae) two-hybrid assays and far-Western studies with purified recombinant ICP35 mapped a core self-association domain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coiled-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-association domain was sufficient to mediate stable self-association only in the presence of additional structural elements in either N- or C-terminal flanking regions. These regions also contain conserved sequences which exhibit a high propensity for alpha helicity and may contribute to self-association by forming additional short coiled coils. Our data supports a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil interactions. In additional two-hybrid experiments we evaluated ICP35 mutants for association with VP5. We discovered that in addition to the C-terminal 25 amino acids of ICP35, previously shown to be required for VP5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domain. Site-directed mutations and construction of chimeric molecules in which the self-association domain of ICP35 was replaced by the GCN4 leucine zipper indicated that this region contributes to VP5 binding through mediating self-association of ICP35 and not through direct binding interactions. Our results suggest that self-association of ICP35 strongly promotes stable association with VP5 in vivo and are consistent with capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.     

Expression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione S-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis.     

Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein.

Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fusion proteins were then incubated with 35S-labeled herpes simplex virus type 1-infected cell lysates containing VP5. The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads. Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C-terminal domain alone is sufficient for interaction with VP5. Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif. Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one. A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong alpha-helix structure. Most interestingly, the alpha-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the alpha-helix. Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction. Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5. In addition, a cyclic analog of the minimal peptide which is designed to stabilize an alpha-helical structure competed more efficiently than the minimal peptide. The evidence suggests that the C-terminal end of ICP35 forms an alpha-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5.     

Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly

Human polyomavirus JC (JCV) can encode the three capsid proteins VP1, VP2, and VP3, downstream of the agnoprotein in the late region. JCV virions are identified in the nucleus of infected cells. In this study, we have elucidated unique features of JCV capsid formation by using a eukaryotic expression system. Structures of JCV polycistronic late RNAs (M1 to M4 and possibly M5 and M6) generated by alternative splicing were determined. VP1 would be synthesized from M2 RNA, and VP2 and VP3 would be synthesized from M1 RNA. The presence of the open reading frame of the agnoprotein or the leader sequence (nucleotides 275 to 409) can decrease the expression level of VP1. VP1 was efficiently transported to the nucleus in the presence of VP2 and VP3 but distributed both in the cytoplasm and in the nucleus in their absence. Mutation analysis indicated that inefficiency in nuclear transport of VP1 is due to the unique structure in the N-terminal sequence, KRKGERK. Within the nucleus, VP1 was localized discretely and identified as speckles in the presence of VP2 and VP3 but distributed diffusely in their absence. These results suggest that VP1 was efficiently transported to the nucleus and localized in the discrete subnuclear regions, possibly with VP2 and VP3. By electron microscopy, recombinant virus particles were identified in the nucleus, and their intranuclear distribution was consistent with distribution of speckles. This system provides a useful model with which to understand JCV capsid formation and the structures and functions of the JCV capsid proteins.     

Molecular interactions of Epstein-Barr virus capsid proteins

The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S-transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.     

Structure of Double-Shelled Rice Dwarf Virus

Rice dwarf virus (RDV), a member of the Reoviridae family, is a double-stranded RNA virus. Infection of rice plants with RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-Å three-dimensional structure of the 700-Å-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1 icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural comparisons were made between the RDV outer shell trimer and the two crystal conformations (REF and HEX) of the VP7 trimer of bluetongue virus, an animal analog of RDV. The low-resolution structural match of the RDV outer shell trimer to the HEX conformation of VP7 trimer has led to the proposal that P8 consists of an upper domain of β-sandwich motif and a lower domain of α helices. The less well fit REF conformation of VP7 to the RDV trimer may be due to the differences between VP7 and P8 in the sequence of the hinge region that connects the two domains. The additional mass density and the absence of a known signaling peptide on the surface of the RDV outer shell trimer may be responsible for the different interactions between plants and animal reoviruses.     

Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion

The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn(2+) ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.     

Identification of an essential domain in the herpesvirus VP1/2 tegument protein: the carboxy terminus directs incorporation into capsid assemblons

The herpesvirus tegument is a layer of viral and cellular proteins located between the capsid and envelope of the virion. The VP1/2 tegument protein is critical for the propagation of all herpesviruses examined. Using an infectious clone of the alphaherpesvirus pseudorabies virus, we have made a collection of truncation and in-frame deletion mutations within the VP1/2 gene (UL36) and examined the resulting viruses for spread between cells. We found that the majority of the VP1/2 protein either was essential for virus propagation or did not tolerate large deletions. A recently described amino-terminal deubiquitinase-encoding domain was dispensable for alphaherpesvirus propagation, but the rate of propagation in an epithelial cell line and the frequency of transport in axons of primary sensory neurons were both reduced. We mapped one essential domain to a conserved sequence at the VP1/2 carboxy terminus and demonstrated that this domain sufficient to redirect the green fluorescent protein to capsid assemblons in nuclei of infected cells.     

Surface loop dynamics in adeno-associated virus capsid assembly

The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.     

Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3

The capsid of SV40 is regarded as a potential nano-capsule for delivery of biologically active materials. The SV40 capsid is composed of 72 pentamers of the VP1 major capsid protein and 72 copies of the minor coat proteins VP2/3. We have previously demonstrated that, when expressed in insect Sf9 cells by the baculovirus system, VP1 self-assembles into virus-like particles (VP1-VLPs), which are morphologically indistinguishable from the SV40 virion and can be easily purified. Here, we show that heterologous proteins fused to VP2/3 can be efficiently incorporated into the VP1-VLPs. Using EGFP as a model protein, we have optimized this encapsulation system and found that fusion to the C-terminus of VP2/3 is preferable and that the C-terminal VP1-interaction domain of VP2/3 is sufficient for incorporation into VLPs. The VLPs encapsulating EGFP retain the ability to attach to the cell surface and enter the cells. Using this system, we have encapsulated yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that converts 5-fluorocytosine to 5-fluorouracil, into VLPs. When CV-1 cells are challenged by the yCD-encapsulating VLPs, they become sensitive to 5-fluorocytosine-induced cell death. Therefore, proteins of interest can be encapsulated in VP1-VLPs by fusion to VP2/3 and successfully delivered to cells.     

Residues of VP26 of herpes simplex virus type 1 that are required for its interaction with capsids

VP26 is the smallest capsid protein and decorates the outer surface of the capsid shell of herpes simplex virus. It is located on the hexons at equimolar amounts with VP5. Its small size (112 amino acids) and high copy number make it an attractive molecule to use as a probe to investigate the complex pattern of capsid protein interactions. An in vitro capsid binding assay and a green fluorescent protein (GFP) localization assay were used to identify VP26 residues important for its interaction with capsids. To test for regions of VP26 that may be essential for binding to capsids, three small in-frame deletion mutations were generated in VP26, Delta18-25, Delta54-60, and Delta93-100. Their designations refer to the amino acids deleted by the mutation. The mutation at the C terminus of the molecule, which encompasses a region of highly conserved residues, abolished binding to the capsid and the localization of GFP to the nucleus in characteristic large puncta. Additional mutations revealed that a region of VP26 spanning from residue 50 to 112 was sufficient for the localization of the fused protein (VP26-GFP) to the nucleus and for it to bind to capsids. Using site-directed mutagenesis of conserved residues in VP26, two key residues for protein-protein interaction, F79 and G93, were identified as judged by the localization of GFP to nuclear puncta. When these mutations were analyzed in the capsid binding assay, they were also found to eliminate binding of VP26 to the capsid structure. Surprisingly, additional mutations that affected the ability of VP26 to bind to capsids in vitro were uncovered. Mutations at residues A58 and L64 resulted in a reduced ability of VP26 to bind to capsids. Mutation of the hydrophobic residues M78 and A80, which are adjacent to the hydrophobic residue F79, abolished VP26 capsid binding. In addition, the block of conserved amino acids in the carboxy end of the molecule had the most profound effect on the ability of VP26 to interact with capsids. Mutation of amino acid G93, L94, R95, R96, or T97 resulted in a greatly diminished ability of VP26 to bind capsids. Yet, all of these residues other than G93 were able to efficiently translocate or concentrate GFP into the nucleus, giving rise to the punctate fluorescence. Thus, the interaction of VP26 with the capsid appears to occur through at least two separate mechanisms. The initial interaction of VP26 and VP5 may occur in the cytoplasm or when VP5 is localized in the nucleus. Residues F79 and G93 are important for this bi-molecular interaction, resulting in the accumulation of VP26 in the nucleus in concentrated foci. Subsequent to this association, additional amino acids of VP26, including those in the C-terminal conserved domain, are important for interaction of VP26 with the three-dimensional capsid structure.     

Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography

Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.     

Refinement of protein structures by iterative comparative modeling and CryoEM density fitting

We developed a method for structure characterization of assembly components by iterative comparative protein structure modeling and fitting into cryo-electron microscopy (cryoEM) density maps. Specifically, we calculate a comparative model of a given component by considering many alternative alignments between the target sequence and a related template structure while optimizing the fit of a model into the corresponding density map. The method relies on the previously developed Moulder protocol that iterates over alignment, model building, and model assessment. The protocol was benchmarked using 20 varied target-template pairs of known structures with less than 30% sequence identity and corresponding simulated density maps at resolutions from 5A to 25A. Relative to the models based on the best existing sequence profile alignment methods, the percentage of C(alpha) atoms that are within 5A of the corresponding C(alpha) atoms in the superposed native structure increases on average from 52% to 66%, which is half-way between the starting models and the models from the best possible alignments (82%). The test also reveals that despite the improvements in the accuracy of the fitness function, this function is still the bottleneck in reducing the remaining errors. To demonstrate the usefulness of the protocol, we applied it to the upper domain of the P8 capsid protein of rice dwarf virus that has been studied by cryoEM at 6.8A. The C(alpha) root-mean-square deviation of the model based on the remotely related template, bluetongue virus VP7, improved from 8.7A to 6.0A, while the best possible model has a C(alpha) RMSD value of 5.3A. Moreover, the resulting model fits better into the cryoEM density map than the initial template structure. The method is being implemented in our program MODELLER for protein structure modeling by satisfaction of spatial restraints and will be applicable to the rapidly increasing number of cryoEM density maps of macromolecular assemblies.     

Ab initio modeling of the herpesvirus VP26 core domain assessed by CryoEM density

Efforts in structural biology have targeted the systematic determination of all protein structures through experimental determination or modeling. In recent years, 3-D electron cryomicroscopy (cryoEM) has assumed an increasingly important role in determining the structures of these large macromolecular assemblies to intermediate resolutions (6–10 Å). While these structures provide a snapshot of the assembly and its components in well-defined functional states, the resolution limits the ability to build accurate structural models. In contrast, sequence-based modeling techniques are capable of producing relatively robust structural models for isolated proteins or domains. In this work, we developed and applied a hybrid modeling approach, utilizing cryoEM density and ab initio modeling to produce a structural model for the core domain of a herpesvirus structural protein, VP26. Specifically, this method, first tested on simulated data, utilizes the cryoEM density map as a geometrical constraint in identifying the most native-like models from a gallery of models generated by ab initio modeling. The resulting model for the core domain of VP26, based on the 8.5-Å resolution herpes simplex virus type 1 (HSV-1) capsid cryoEM structure and mutational data, exhibited a novel fold. Additionally, the core domain of VP26 appeared to have a complementary interface to the known upper-domain structure of VP5, its cognate binding partner. While this new model provides for a better understanding of the assembly and interactions of VP26 in HSV-1, the approach itself may have broader applications in modeling the components of large macromolecular assemblies.     

Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid. A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5). Previously (Z. Hong, M. Beaudet-Miller, J. Durkin, R. Zhang, and A. D. Kwong, J. Virol. 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix. The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction. Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein. This allowed us to focus our efforts on a small region of this large polypeptide. Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine. All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay. From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified. All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system. For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made. The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses. The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented. Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure. Thus, it appears that the L75 residue is important for another essential interaction of VP5 with the capsid shell proteins. The congruence of the data from the previous and present studies demonstrates the key roles of two regions in the N terminus of this large protein that are crucial for this bimolecular interaction. Thus, residues I27, L35, and F39 comprise the first subdomain and residues L58, L65, L67 and L71 comprise a second subdomain of VP5. These seven hydrophobic residues are important for the interaction of VP5 with the scaffold protein and consequently the formation of an icosahedral shell structure that encloses the viral genome.