Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships

Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses.     

A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids

Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as S. aureus pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T = 7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97 and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue β hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phage HK97 or P22.     

An elastic network model of HK97 capsid maturation

The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. Typically the capsid assembles through polymerization and maturation processes. Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II. We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II. The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer. We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints. Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II. The feasibility of the predicted pathway is also supported by the results of our normal mode analysis.     

Bacteriophage lambda stabilization by auxiliary protein gpD: timing, location, and mechanism of attachment determined by cryo-EM

We report the cryo-EM structure of bacteriophage lambda and the mechanism for stabilizing the 20-A-thick capsid containing the dsDNA genome. The crystal structure of the HK97 bacteriophage capsid fits most of the T = 7 lambda particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein gpD, which is necessary for stabilization of the capsid shell. Its position coincides with the location of the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement of this gene product in lambda by autocatalytic chemistry in HK97. The crystal structure of the trimeric gpD, in which the 14 N-terminal residues required for capsid binding are disordered, fits precisely into the corresponding EM density. The N-terminal residues of gpD are well ordered in the cryo-EM density, adding a strand to a beta-sheet formed by the capsid proteins and explaining the mechanism of particle stabilization.     

HK97 Maturation Studied by Crystallography and H/H Exchange Reveals the Structural Basis for Exothermic Particle Transitions

HK97 is an exceptionally amenable system for characterizing major conformational changes associated with capsid maturation in double-stranded DNA bacteriophage. HK97 undergoes a capsid expansion of ∼ 20%, accompanied by major subunit rearrangements during genome packaging. A previous 3.44-Å-resolution crystal structure of the mature capsid Head II and cryo-electron microscopy studies of other intermediate expansion forms of HK97 suggested that, primarily, rigid-body movements facilitated the maturation process. We recently reported a 3.65-Å-resolution structure of the preexpanded particle form Prohead II (P-II) and found that the capsid subunits undergo significant refolding and twisting of the tertiary structure to accommodate expansion. The P-II study focused on major twisting motions in the P-domain and on refolding of the spine helix during the transition. Here we extend the crystallographic comparison between P-II and Head II, characterizing the refolding events occurring in each of the four major domains of the capsid subunit and their effect on quaternary structure stabilization. In addition, hydrogen/deuterium exchange, coupled to mass spectrometry, was used to characterize the structural dynamics of three distinct capsid intermediates: P-II, Expansion Intermediate, and the nearly mature Head I. Differences in the solvent accessibilities of the seven quasi-equivalent capsid subunits, attributed to differences in secondary and quaternary structures, were observed in P-II. Nearly all differences in solvent accessibility among subunits disappear after the first transition to Expansion Intermediate. We show that most of the refolding is coupled to this transformation, an event associated with the transition from asymmetric to symmetric hexamers.     

Time-resolved molecular dynamics of bacteriophage HK97 capsid maturation interpreted by electron cryo-microscopy and X-ray crystallography

The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.     

1H, 15N, and 13C resonance assignments for a monomeric mutant of the HIV-1 capsid protein

The mature fullerene cone-shaped capsid of the human immunodeficiency virus 1 is composed of about 1,500 copies of the capsid protein (CA). The CA is 231 residues long, and consists of two distinct structural domains, the N-terminal domain and the C-terminal domain (CTD), joined by a flexible linker. The wild type CA exhibits monomer-dimer equilibrium in solution through the CTD-CTD dimerization. This CTD-CTD interaction, together with other intermolecular interdomain interactions, plays significant roles during the assembly of the mature capsid. In addition, CA-CA interactions also play a role in the assembly of the immature virion. The CA also interacts with some host cell proteins within the viral replication cycle. Thus, the capsid protein has been of significant interest as a target for designing inhibitors of assembly of immature virions and mature capsids and inhibitors of its interactions with host cell proteins. However, the equilibrium exhibited by the wild-type CA protein between the monomeric and dimeric states, along with the inherent flexibility from the interdomain linker, have hindered attempts at structural determination by solution NMR and X-ray crystallography methods. In this study, we have utilized a CA protein with W184A and M185A mutations that abolish the dimerization of CA protein as well as its infectivity, but preserve most of the remaining properties of the wild type CA. We have determined the detailed solution structure of the monomeric W184A/M185A-CA protein using 3D-NMR spectroscopy. Here, we present the detailed sequence-specific NMR assignments for this protein.     

Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM

Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid, but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described. Here, we report a cryo electron microscopy (cryoEM) structure of the murine cytomegalovirus (MCMV) capsid at a 9.1 ? resolution and describe the molecular interactions among the ~3000 protein molecules in the CMV capsid at the secondary structure level. Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling. The major capsid protein (MCP) upper domain (MCPud) contains α-helices and β-sheets conserved with those in MCPud of herpes simplex virus type 1 (HSV-1), with the largest differences identified as a “saddle loop” region, located at the tip of MCPud and involved in interaction with the smallest capsid protein (SCP). Interactions among the bacteriophage HK97-like floor domain of MCP, the middle domain of MCP, the hook and clamp domains of the triplex proteins (hoop and clamp domains of TRI-1 and clamp domain of TRI-2) contribute to the formation of a mature capsid. These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attaching unique functional tegument proteins outside.     

Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids

The capsid of bacteriophage HK97 is stabilized by ∼400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found ∼2.4 Å from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution.A common feature of the assembly pathways of all tailed double-stranded DNA bacteriophages and herpesviruses (45) is that they package their chromosomes into unstable precursors called procapsids or proheads, which mature into virions. The virion DNA is packed to a very high density (∼0.5 g/ml) (13), so the completed DNA-filled capsids must be specifically reinforced or stabilized during maturation to withstand the very high effective internal “DNA pressure” (20, 41, 49). Virions also must resist the chemical and physical stresses of the environment outside their hosts. Phage capsids achieve this stabilization by several different mechanisms, all of which include large conformational changes in the capsid. Some phages, like P22 (14) and T7 (4), achieve stabilization by conformational changes alone. For other phages, such as T4 (43) and λ (42), full stabilization requires the binding of so-called “decoration” proteins to the outside of the capsid (44). Capsid stabilization may also occur by forming covalent cross-links between capsid protein subunits, as in phage HK97 (10, 11, 36, 51). The HK97 cross-links are isopeptide bonds between asparagine and lysine side chains on neighboring subunits that tie together structural elements on the inside of the shell to elements on the outside, thus generating a chain-mail-like network of connections among capsid subunits and making a very stable capsid (39, 51). This type of amide bond was thought to occur only in phage capsid proteins, where they occur frequently (23), but it has also been identified recently within the subunits of the pili of Streptococcus pyogenes and may be widespread in related pilus proteins (26).HK97 is a λ-like phage of Escherichia coli (7, 25) with a long noncontractile tail and a T=7 icosahedral capsid (5). HK97 procapsids assemble from the major capsid protein, the capsid protease, and the portal protein as outlined in Fig. ​Fig.1.1. Assembly occurs during phage infection or when the same proteins are expressed from plasmids in E. coli. After assembly of Prohead I, the protease cleaves interior domains from the major capsid protein and digests itself, yielding the procapsid, Prohead II, which contains only cleaved major capsid protein. Prohead II is the natural substrate for DNA packaging during phage infection, and it matures during packaging by a process called expansion and transforms into Head II, for which the X-ray structure has been determined (22, 51). Expansion can also be induced chemically in vitro and has been studied extensively, both biochemically and in multiple cryo-electron microscopy (cryo-EM) and X-ray structures (5, 6, 15, 16, 31-33, 39). Expansion involves extensive rearrangements of the capsid subunits which change the round, thick-walled ∼550-Å Prohead II into the angular, thin-walled ∼650Å Head II (Fig. ​(Fig.1).1). Capsid cross-links are made during expansion.Open in a separate windowFIG. 1.Assembly and maturation of the HK97 capsid. Three proteins are required to assemble Prohead II, the HK97 procapsid. These are the major capsid protein (gp5, 42 kDa), the protease (gp4, 25 kDa), and the portal or connector protein (gp3, 46 kDa) (12). The major capsid protein folds and assembles into hexamers and pentamers (capsomers collectively), which coassemble with the protease to form Prohead I. The protease removes 102 residues of each capsid subunit and destroys itself to create Prohead II. The portal complex normally occupies the position of one pentamer and is required for viable phage assembly, but the portal protein is optional for making particles from plasmid-expressed genes (5, 11). Prohead II expands to form the mature capsid, Head II, normally during DNA packaging, but also in vitro as described in Results.The high-resolution structure of the HK97 mature capsid provides a structural basis for understanding many aspects of assembly and cross-linking (22, 51). The subunits of the mature capsid have a continuous core with a dominant long alpha-helix and two extended arms, the N-arm and the long β-hairpin or E-loop (for Extended loop), as shown in Fig. ​Fig.2A.2A. Cross-links cannot form until a complete particle is assembled—this can be easily understood by looking at the Head II subunit by itself and the arrangement of subunits in the mature particle (Fig. 2A and B). The three residues required for cross-linking are far apart in an isolated subunit, as they are in an individual hexamer or pentamer of capsid protein from which the proheads assemble. At least three capsomers (hexamers or pentamers of capsid protein) must assemble in a manner like that shown in Fig. 2B and C in order to bring together the three residues that will participate in a given cross-link. This is not achieved directly; the capsomers initially assemble into proheads, in which the two residues that will cross-link are still separated by more than 30 Å (6). Cross-linking does not occur until the conformational rearrangements of the expansion reaction bring asparagine 356 (N356) and lysine 169 (K169) close to glutamic acid 363 (E363) in an active site-like cavity. We argue here that E363 has a catalytic role in the cross-linking reaction.Open in a separate windowFIG. 2.Three-dimensional models of the sites of capsid cross-linking in HK97. Views of HK97 cross-link sites based on the refined structure (PDB ID 1OHG) (22) show the proximity of the side chain of glutamate E363 to the joined residues. (A) An HK97 Head II subunit (D chain) in isolation shows the three side chains involved in capsid cross-linking: an asparagine (N356, dark blue) at the end of the P-domain, a lysine (K169, red) near the tip of the E-loop, and a glutamate (E363, orange) protruding from a β-sheet (β-J) in the P-domain. HK97 cross-links occur at sites located around the threefold and quasi-threefold axes of symmetry of the HK97 capsid, and one such trio of cross-links is shown in panels B and C. (B) Cross-links formed around a threefold axis (red-filled triangle) and their context among the three hexons that comprise a flat icosahedral face of the particle. The hexamer chains are labeled A to F, and the locations of several quasi-threefold axes are marked with open triangles. Views are from the inside of a capsid, looking outward. (C) Close-up view of the area boxed in panel B. The chains joined by the highlighted cross-links are colored identically in pairs: a lysine from a D chain is joined to an asparagine in an E chain from a neighboring hexamer, aided by a glutamate in the D chain of a neighboring hexamer. Side chain oxygens in the glutamates and cross-links are indicated in red. In the lower of the three cross-link sites, the side chains of three hydrophobic residues that surround the cross-links are shown in space-filling representations; these are residues V163, L361, and M339 and are labeled in red. Images made using SPDBV 3.7 (21) and the Persistence of Vision Raytracer (POV-Ray Mac 3.6 [http://www.povray.org/]).In the Head II X-ray model (22), one of the cross-link residues, N356, is near the outer end of the P-domain, sandwiched within the subunit interface near junctions where three hexamers (or two hexamers and a pentamer) meet (see Fig. ​Fig.2),2), while the other, K169, projects into the subunit interface from near the tip of the E-loop. The third residue involved in cross-linking, E363, is located on a third subunit, which lies across the local capsomer-capsomer interface from N356 (Fig. 2B and C) and one carboxylate oxygen of E363 is found only 2.5 Å away from the isopeptide bond. This arrangement is found in each of the seven unique (quasi-equivalent) cross-link sites in the Head II model (22, 51). Changing E363 to alanine (E363A) was found to abolish cross-linking (51), suggesting that E363 may have an important chemical role in the cross-linking reaction. Further support for a role of E363 in cross-linking comes from evidence that it appears to be conserved and easily identified in alignments of the HK97 capsid protein sequence with other capsid proteins in GenBank. For example, a BLAST search of the nonredundant protein database with the mature HK97 capsid protein sequence yields ∼60 non-self-matches, and 43 of these potential homologs appear to possess all three cross-linking residues (matching K169, N356, and E363 in HK97), notably including several bacteriophages for which there is experimental evidence of capsid cross-linking: D3 (19), XP10 (54), BFK20 (3), and Φ11b (2). The findings we present here suggest that both the size and the character of residue 363 can influence assembly and maturation of HK97 capsids, but only glutamate is capable of promoting efficient cross-linking. In the analogous Asn-Lys isopeptide bonds recently identified in the pili of gram-positive bacteria, a conserved glutamic (or aspartic) acid residue is also poised nearby and implicated in cross-linking, suggesting a common mechanism.     

The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174.

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.     

Andersen's syndrome mutation effects on the structure and assembly of the cytoplasmic domains of Kir2.1

Kir2.1 channels play a key role in maintaining the correct resting potential in eukaryotic cells. Recently, specific amino acid mutations in the Kir2.1 inwardly rectifying potassium channel have been found to cause Andersen's Syndrome in humans. Here, we have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and delta314-315 and have found multiple effects on the ability of the cytoplasmic domains in Kir2.1 channels to form proper tetrameric assemblies. For the R218Q mutation, we identified a second site mutation (T309K) that restored tetrameric assembly but not function. We successfully crystallized and solved the structure (at 2.0 A) of the N- and C-terminal cytoplasmic domains of Kir2.1-R218Q/T309K(S). This new structure revealed multiple conformations of the G-loop and CD loop, providing an explanation for channels that assemble but do not conduct ions. Interestingly, Glu303 forms both intra- and intersubunit salt bridges, depending on the conformation of the G-loop, suggesting that the E303K mutant stabilizes both closed and open G-loop conformations. In the Kir2.1-R218Q/T309K(S) structure, we discovered that the DE loop forms a hydrophobic pocket that binds 2-methyl-2,4-pentanediol, which is located near the putative G(betagamma)-activation site of Kir3 channels. Finally, we observed a potassium ion bound to the cytoplasmic domain for this class of K+ channels.     

Critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly

During retroviral maturation, the CA protein undergoes dramatic structural changes and establishes unique intermolecular interfaces in the mature capsid shell that are different from those that existed in the immature precursor. The most conserved region of CA, the major homology region (MHR), has been implicated in both immature and mature assembly, although the precise contribution of the MHR residues to each event has been largely undefined. To test the roles of specific MHR residues in mature capsid assembly, an in vitro system was developed that allowed for the first-time formation of Rous sarcoma virus CA into structures resembling authentic capsids. The ability of CA to assemble organized structures was destroyed by substitutions of two conserved hydrophobic MHR residues and restored by second-site suppressors, demonstrating that these MHR residues are required for the proper assembly of mature capsids in addition to any role that these amino acids may play in immature particle assembly. The defect caused by the MHR mutations was identified as an early step in the capsid assembly process. The results provide strong evidence for a model in which the hydrophobic residues of the MHR control a conformational reorganization of CA that is needed to initiate capsid assembly and suggest that the formation of an interdomain interaction occurs early during maturation.     

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.     

Quasi-atomic model of bacteriophage t7 procapsid shell: insights into the structure and evolution of a basic fold

The existence of similar folds among major structural subunits of viral capsids has shown unexpected evolutionary relationships suggesting common origins irrespective of the capsids' host life domain. Tailed bacteriophages are emerging as one such family, and we have studied the possible existence of the HK97-like fold in bacteriophage T7. The procapsid structure at approximately 10 A resolution was used to obtain a quasi-atomic model by fitting a homology model of the T7 capsid protein gp10 that was based on the atomic structure of the HK97 capsid protein. A number of fold similarities, such as the fitting of domains A and P into the L-shaped procapsid subunit, are evident between both viral systems. A different feature is related to the presence of the amino-terminal domain of gp10 found at the inner surface of the capsid that might play an important role in the interaction of capsid and scaffolding proteins.     

Intragenic suppressors of an OmpF assembly mutant and assessment of the roles of various OmpF residues in assembly through informational suppressors

We employed two separate genetic approaches to examine the roles of various OmpF residues in assembly. In one approach, intragenic suppressors of a temperature-sensitive OmpF assembly mutant carrying a W214E substitution were sought at 42 degrees C, or at 37 degrees C in a genetic background lacking the periplasmic folding factor SurA. In the majority of cases (58 out of 61 revertants), the suppressors mapped either at the original site (position 214) or two residues downstream from it. In the remaining three revertants that were obtained in a surA background, an alteration of N230Y was located 16 residues away from the original site. The N230Y suppressor also corrected OmpF315 assembly at 42 degrees C in a surA(+) background, indicating that the two different physiological environments imposed similar assembly constraints. The specificity of N230Y was tested against five different residues at position 214 of mature OmpF. Clear specificity was displayed, with maximum suppression observed for the original substitution at position 214 (E214) against which the N230Y suppressor was isolated, and no negative effect on OmpF assembly was noted when the wild-type W214 residue was present. The mechanism of suppression may involve compensation for a specific conformational defect. The second approach involved the application of informational suppressors (Su-tRNA) in combination with ompF amber mutations to generate variant OmpF proteins. In this approach we targeted the Y40, Q66, W214, and Y231 residues of mature OmpF and replaced them with S, Q, L, and Y through the action of Su-tRNAs. Thus, a total of 16 variant OmpF proteins were generated, of which three were identical to the parental protein, and two variants carrying W214Q and Y231Q substitutions were similar to assembly-defective proteins isolated previously (R. Misra, J. Bacteriol. 175:5049-5056, 1993). The results obtained from these analyses provided useful information regarding the compatibility of various alterations in OmpF assembly.     

Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability.

In the spherical capsid of hepatitis B virus (HBV), intermolecular disulfide bonds cross-link the approximately 180 p21.5 capsid protein subunits into a stable lattice. In this study, we used mutant capsid proteins to investigate the role that disulfide bonds and the four p21.5 Cys residues (positions 48, 61, 107, and 185) play in capsid assembly and/or stabilization. p21.5 Cys residues were either replaced by Ala or removed (Cys-185) by carboxyl-terminal truncation, creating Cys-minus mutants which were expressed in Xenopus oocytes via microinjected synthetic mRNAs. Fractionation of radiolabeled oocyte extracts on 10 to 60% sucrose gradients revealed that Cys-minus core proteins resolved into the nonparticulate and capsid forms seen for wild-type p21.5. On 5 to 30% sucrose gradients, nonparticulate Cys-minus core proteins sedimented as dimers of approximately 40 kDa. We conclude that Cys residues and disulfides are not required for the assembly of either HBV capsids or the dimers that provide the precursors for capsid assembly. Since assembly presumably demands an appropriate p21.5 tertiary structure, it is unlikely that Cys residues are required for proper p21.5 folding. However, Cys residues stabilize isolated p21.5 structures, as evidenced by the marked reduction in stability of Cys-minus dimers and capsids (i) in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and (ii) upon protease digestion. We discuss these results in the context of the HBV life cycle and the role of Cys residues in other proteins.     

Structural tolerance versus functional intolerance to mutation of hydrophobic core residues surrounding cavities in a parvovirus capsid

The structural and functional relevance of amino acid residues surrounding cavities within the hydrophobic core of the protein subunits that form the capsid of parvoviruses has been investigated. Several of the evolutionarily conserved, hydrophobic residues that delimit these cavities in the capsid of the minute virus of mice were replaced by other hydrophobic residues that would affect the size and/or shape of the cavity. When four or more methylene-sized groups were introduced, or six or more groups removed, capsid assembly was drastically impaired. In contrast, the introduction or removal of up to three groups had no significant effect on capsid assembly or thermostability. However, many of these mutations affected a capsid conformational transition needed for viral infectivity. Replacement of some polar residues around the largest cavity showed that capsid assembly requires a carboxylate buried within this cavity, but both aspartate and glutamate are structurally accepted. Again, only the aspartate allowed the production of infectious viruses, because of a specific role in encapsidation of the viral genome. These observations provide evidence of a remarkable structural tolerance to mutation of the hydrophobic core of the protein subunits in a viral capsid, and of an involvement of core residues and internal cavities in capsid functions needed for infectivity.     

A dissection of the protein-protein interfaces in icosahedral virus capsids

We selected 49 icosahedral virus capsids whose crystal structures are reported in the Protein Data Bank. They belong to the T=1, T=3, pseudo T=3 and other lattice types. We identified in them 779 unique interfaces between pairs of subunits, all repeated by icosahedral symmetry. We analyzed the geometric and physical chemical properties of these interfaces and compared with interfaces in protein-protein complexes and homodimeric proteins, and with crystal packing contacts. The capsids contain one to 16 subunits implicated in three to 66 unique interfaces. Each subunit loses 40-60% of its accessible surface in contacts with an average of 8.5 neighbors. Many of the interfaces are very large with a buried surface area (BSA) that can exceed 10,000 A(2), yet 39% are small with a BSA<800 A(2) comparable to crystal packing contacts. Pairwise capsid interfaces overlap, so that one-third of the residues are part of more than one interface. Those with a BSA>800 A(2) resemble homodimer interfaces in their chemical composition. Relative to the protein surface, they are non-polar, enriched in aliphatic residues and depleted of charged residues, but not of neutral polar residues. They contain one H-bond per about 200 A(2) BSA. Small capsid interfaces (BSA<800 A(2)) are only slightly more polar. They have a similar amino acid composition, but they bury fewer atoms and contain fewer H-bonds for their size. Geometric parameters that estimate the quality of the atomic packing suggest that the small capsid interfaces are loosely packed like crystal packing contacts, whereas the larger interfaces are close-packed as in protein-protein complexes and homodimers. We discuss implications of these findings on the mechanism of capsid assembly, assuming that the larger interfaces form first to yield stable oligomeric species (capsomeres), and that medium-size interfaces allow the stepwise addition of capsomeres to build larger intermediates.     

Molecular dissection of ø29 scaffolding protein function in an in vitro assembly system

An in vitro assembly system was developed to study prolate capsid assembly of phage ?29 biochemically, and to identify regions of scaffolding protein required for its functions. The crowding agent polyethylene glycol can induce bacteriophage ?29 monomeric capsid protein and dimeric scaffolding protein to co-assemble to form particles which have the same geometry as either prolate T=3 Q=5 procapsids formed in vivo or previously observed isometric particles. The formation of particles is a scaffolding-dependent reaction. The balance between the fidelity and efficiency of assembly is controlled by the concentration of crowding agent and temperature. The assembly process is salt sensitive, suggesting that the interactions between the scaffolding and coat proteins are electrostatic. Three N-terminal ?29 scaffolding protein deletion mutants, Delta 1-9, Delta 1-15 and Delta 1-22, abolish the assembly activity. Circular dichroism spectra indicate that these N-terminal deletions are accompanied by a loss of helicity. The inability of these proteins to dimerize suggests that the N-terminal region of the scaffolding protein contributes to the dimer interface and maintains the structural integrity of the dimeric protein. Two C-terminal scaffolding protein deletion mutants, Delta 79-97 and Delta 62-97, also fail to promote assembly. However, the secondary structure and the dimerization ability of these mutants are unchanged relative to wild-type, which suggests that the C terminus is the likely site of interaction with the capsid protein.     

L1 interaction domains of papillomavirus l2 necessary for viral genome encapsidation

BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.