Encapsulins are bacterial organelle-like cages involved in various aspects of metabolism, especially protection from oxidative stress. They can serve as vehicles for a wide range of medical applications. Encapsulin shell proteins are structurally similar to HK97 bacteriophage capsid protein and their function depends on the encapsulated cargos. The Myxococcus xanthus encapsulin system comprises EncA and three cargos: EncB, EncC, and EncD. EncB and EncC are similar to bacterial ferritins that can oxidize Fe+2 to less toxic Fe+3. We analyzed EncA, EncB, and EncC by cryo-EM and X-ray crystallography. Cryo-EM shows that EncA cages can have T = 3 and T = 1 symmetry and that EncA T = 1 has a unique protomer arrangement. Also, we define EncB and EncC binding sites on EncA. X-ray crystallography of EncB and EncC reveals conformational changes at the ferroxidase center and additional metal binding sites, suggesting a mechanism for Fe oxidation and storage within the encapsulin shell.

Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures.

INI1/SMARCB1 binds to HIV-1 integrase (IN) through its Rpt1 domain and exhibits multifaceted role in HIV-1 replication. Determining the NMR structure of INI1-Rpt1 and modeling its interaction with the IN-C-terminal domain (IN-CTD) reveal that INI1-Rpt1/IN-CTD interface residues overlap with those required for IN/RNA interaction. Mutational analyses validate our model and indicate that the same IN residues are involved in both INI1 and RNA binding. INI1-Rpt1 and TAR RNA compete with each other for IN binding with similar IC50 values. INI1-interaction-defective IN mutant viruses are impaired for incorporation of INI1 into virions and for particle morphogenesis. Computational modeling of IN-CTD/TAR complex indicates that the TAR interface phosphates overlap with negatively charged surface residues of INI1-Rpt1 in three-dimensional space, suggesting that INI1-Rpt1 domain structurally mimics TAR. This possible mimicry between INI1-Rpt1 and TAR explains the mechanism by which INI1/SMARCB1 influences HIV-1 late events and suggests additional strategies to inhibit HIV-1 replication. HIV-1 integrase (IN) binds the host factor INI1/SMARCB1, which is required at multiple stages of HIV-1 replication. Here, the authors show that the same IN residues are involved in INI1 and RNA binding and in influencing particle morphogenesis and suggest that the IN-binding INI1 domain is structurally similar to HIV TAR RNA.

1 All structures are T = 4 capsids except 3KXS, which is a core-antigen dimer complex. All structures are apo, i.e. non-liganded. (DOCX)

1 All the chain pairs in the table were analyzed with DynDom. However, when the chain pairs did not meet the specific requirements of being “dynamic”, even if their conformations were different, then no values were returned by the program and the cells were left blank. 2 Indicates that chains from T = 3 capsids were compared to chains from T = 4 capsids, in the order given. (DOCX)

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

1 Only human viral structures are listed, i.e. no WHV, although the piscine T = 3 Nackednaviral structures are included as they are considered in this study. Also, only core-antigen structures are included, i.e. no e-antigen. 2 3J2V, 6BVF, and 6BVN are in both databases but are only shown once here, in the PDB. 3 Symmetry; NA indicates a non-capsid complex. 4 When no resolution was reported (-). (DOCX)

Hepatitis B virus (HBV) is a leading cause of liver disease. The capsid is an essential component of the virion and it is therefore of interest how it assembles and disassembles. The capsid protein is unusual both for its rare fold and that it polymerizes according to two different icosahedral symmetries, causing the polypeptide chain to exist in seven quasi-equivalent environments: A, B, and C in AB and CC dimers in T = 3 capsids, and A, B, C, and D in AB and CD dimers in T = 4 capsids. We have compared the two capsids by cryo-EM at 3.5 Å resolution. To ensure a valid comparison, the two capsids were prepared and imaged under identical conditions. We find that the chains have different conformations and potential energies, with the T = 3 C chain having the lowest. Three of the four quasi-equivalent dimers are asymmetric with respect to conformation and potential energy; however, the T = 3 CC dimer is symmetrical and has the lowest potential energy although its intra-dimer interface has the least free energy of formation. Of all the inter-dimer interfaces, the CB interface has the least area and free energy, in both capsids. From the calculated energies of higher-order groupings of dimers discernible in the lattices we predict early assembly intermediates, and indeed we observe such structures by negative stain EM of in vitro assembly reactions. By sequence analysis and computational alanine scanning we identify key residues and motifs involved in capsid assembly. Our results explain several previously reported observations on capsid assembly, disassembly, and dimorphism.

The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins (“ejection proteins”) have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions; it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100–110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.

1 Results from a KFC2 computational alanine scanning interface analysis of the four quasi-equivalent sites (AA, BD, CB, and DC) in the T = 4 capsids described in this study. Residues identified as hot spots by KFC2 are listed separately for each chain in a dimer. Residues in bold font were classed as high confidence by KFC2. Area and volume values are for the interface region between the two chains, 3 A from each chain, as calculated with FADE (on the KFC2 server) and UCSF Chimera. Note that the CB interface is smaller than the other three and that V124 and R127 are not classed as part of the CB interface (boxed cells). (DOCX)

“Giant” phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them “mottled capsids”. Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a “twisted hook” conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.

1 Results from a KFC2 computational alanine scanning interface analysis of the three quasi-equivalent sites (AA, BC, and CB) in the T = 3 capsids described in this study. Residues identified as hot spots by KFC2 are listed separately for each chain in a dimer. Residues in bold font were classed as high confidence by KFC2. Area and volume values are for the interface region between the two chains, 3 A from each chain, as calculated with FADE (on the KFC2 server) and UCSF Chimera. Note that the CB interface is smaller and that V124 and R127 are not classed as part of the CB interface (boxed cells). (DOCX)

This table is similar to Table 1 but is more inclusive. 1, 2 All score values are percent (%) except Clash score and Molprobity score, which are percentile, as defined below. 3 Clash score is the number of serious steric overlaps (>0.4 A) per 1,000 atoms. 4 Molprobity score combines clash, rotamer, and Ramachandran evaluations into a single score, normalized to be on the same scale as X-ray resolution. For both Clash score and Molprobity score the values are percentile (100th is best, 0th is worst) relative to a set of comparable structures determined for each calculation (see Molprobity server for details). Analysis performed with PROCHECK confirmed the quality of the current structures. (DOCX)

The hepatitis B virus e antigen, an alternative transcript of the core gene, is a secreted protein that maintains viral persistence. The physiological form has extended C termini relative to Cp(‐10)149, the construct used in many studies. To examine the role of the C termini, we expressed the constructs Cp(‐10)151 and Cp(‐10)154, which have additional arginine residues. Both constructs when treated with reductant formed capsids more efficiently than Cp(‐10)149. These capsids were also substantially more stable, as measured by thermal denaturation and resistance to urea dissociation. Mutagenesis suggests that electrostatic interactions between the additional arginine residues and glutamate residues on adjacent subunits play a role in the extra stabilization. These findings have implications for the physiological role and biotechnological potential of this protein.

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

In studying biologically relevant molecules, the ultimate aim is to visualize them in their native environment. One technique that has the potential to resolve individual molecules in cells and tissues is cryo-electron microscopy of vitreous sections (CEMOVIS) combined with tomography. We are interested in revealing the disposition of the retinal adhesion protein, retinoschisin (RS1), within the retina. Immuno-fluorescence and immuno-electron microscopy showed that RS1 is abundant in the intercellular spaces between the inner segments of photoreceptors, and between bipolar cells. Loss-of-function mutations in RS1 cause X-linked retinoschisis (XLRS), a debilitating disease associated with the formation of retinal cavities or cysts (schisis) and vision loss in young males. We solved the structure to 4.1 Å by cryo-electron microscopy (cryoEM), a 16-mer composed of two octameric rings with a total weight of ∼380 kDa. From this structure it is clear that many mutations in the subunit interfaces would preclude assembly of the rings. However, other mutation sites are exposed at the periphery and it was not evident how they would cause disease. In further cryoEM experiments, we observed that RS1 double rings self-assemble into extensive branched networks, suggesting a mechanism for its adhesive function. To ascertain whether this assembly also occurs in situ, we are examining mouse retinal tissue using CEMOVIS and cryo-electron tomography. We obtained images of 50 nm cryosections that show clear distinction of microtubular protofilaments separated by ∼5 nm. This resolution is clearly sufficient to see the RS1 double ring and whether it assembles into networks in the intercellular space between photoreceptors and bipolar cells.