Replication-defective vector based on a chimpanzee adenovirus.

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.

Type-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexon.

A major obstacle to the use of adenovirus as a vector for gene therapy is the host immune response to hexon, the major protein component of the icosahedral capsid. A solution lies in creating novel vectors with modified or chimeric hexons to evade the immune response to native hexon. The crystal structure of hexon from human adenovirus type 5 (ad5), the type primarily used for gene therapy, has been determined to facilitate the design of such molecules. As the 951-amino-acid (aa) ad5 hexon sequence is closely related to that of ad2 (967 aa; 86% aa identity), the ad5 structure was solved by molecular replacement with a model constructed from the known ad2 hexon. During refinement, greater than 25% of the sequence was reassigned, resulting in a relocation of two epitope regions, from buried positions in the ad2 model to external sites at the top of the ad5 molecule. The resultant model is in better agreement with crystallographic data, while maintaining the overall topology of ad2 hexon. This work suggests that all hexons have the same basic fold and that the ad5 hexon structure provides an accurate and representative model for designing new adenovirus vectors.

Spherical viruses.

In the past two years, structural studies on spherical viruses have experienced a significant advance with the dramatic increase in the resolution attainable by cryo-electron microscopy and image reconstruction. X-ray crystallography, both alone and, increasingly, in combination with electron microscopy, continues to play a crucial role in elucidating how viruses function.

Probing the heme iron coordination structure of pressure-induced cytochrome P420cam.

Cytochrome P450cam was subjected to high pressures of 2.2 kbar, converting the enzyme to its inactive form P420cam. The resultant protein was characterized by electron paramagnetic resonance, magnetic circular dichroism, circular dichroism, and electronic absorption spectroscopy. A range of exogenous ligands has been employed to probe the coordination structure of P420cam. The results suggest that conversion to P420cam involves a conformational change which restricts the substrate binding site and/or alters the ligand access channel. The reduction potential of P420cam is essentially the same in the presence or absence of camphor (-211 +/- 10 and -210 +/- 15 mV, respectively). Thus, the well-documented thermodynamic regulation of enzymatic activity for P450cam in which the reduction potential is coupled to camphor binding is not found with P420cam. Further, cyanide binds more tightly to P420cam (Kd = 1.1 +/- 0.1 mM) than to P450cam (Kd = 4.6 +/- 0.2 mM), reflecting a weakened iron-sulfur ligation. Spectral evidence reported herein for P420cam as well as results from a parallel investigation of the spectroscopically related inactive form of chloroperoxidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420cam.

Probing the heme iron coordination structure of alkaline chloroperoxidase.

The mechanism by which the heme-containing peroxidase, chloroperoxidase, is able to chlorinate substrates is poorly understood. One approach to advance our understanding of the mechanism of the enzyme is to determine those factors which contribute to its stability. In particular, under alkaline conditions, chloroperoxidase undergoes a transition to a new, spectrally distinct form, with accompanying loss of enzymatic activity. In the present investigation, ferric and ferrous alkaline chloroperoxidase (C420) have been characterized by electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy. The heme iron oxidation state influences the transition to C420; the pKa for the alkaline transition is 7.5 for the ferric protein and 9.5 for the ferrous protein. The five-coordinate, high-spin ferric native protein converts to a six-coordinate low-spin species (C420) as the pH is raised above 7.5. The inability of ferric C420 to bind exogenous ligands, as well as the dramatically increased reactivity of the proximal Cys29 heme ligand toward modification by the sulfhydryl reagent p-mercuribenzoate, suggests that a conformational change has occurred during conversion to C420 that restricts access to the peroxide binding site while increasing the accessibility of Cys29. However, it does appear that Cys29-derived ligation is at least partially retained by ferric C420, potentially in a thiolate/imidazole coordination sphere. Ferrous C420, on the other hand, appears not to possess a thiolate ligand but instead likely has a bis-imidazole (histidine) coordination structure. The axial ligand trans to carbon monoxide in ferrous-CO C420 may be a histidine imidazole. Since chloroperoxidase functions normally through the ferric and higher oxidation states, the fact that the proximal thiolate ligand is largely retained in ferric C420 clearly indicates that additional factors such as the absence of a vacant sixth coordination site sufficiently accessible for peroxide binding may be the cause of catalytic inactivity.

Crystallization and preliminary X-ray analysis of herpes simplex virus neutralizing antibody Fab fragment DL11.

An Fab fragment of a virus-neutralizing monoclonal antibody (DL11) that binds to herpes simplex virus glycoprotein D (HSV gD) has been purified, sequenced and crystallized. The biological activity of the purified Fab was verified by enzyme-linked immunosorbant assay, flow cytometry and by neutralization of HSV infectivity. The crystals have the space group P1 with cell dimensions a = 40.2, b = 49.2, c = 63.9 A, alpha = 76.1, beta = 77.4, gamma = 71.6 degrees. The unit-cell volume is consistent with it containing a single Fab molecule. The crystals grow to a maximum size of 0.7 x 0.3 x 0.3 mm and diffract X-rays to greater than 2.2 A resolution. The amino-acid sequences of the variable regions of the heavy and light chains of DL11 have been determined. These have been compared to those for other known Fab structures in the Protein Data Bank for selection of a starting model for crystallographic refinement by the molecular-replacement method.

The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 A resolution.

The crystal structure of hexon, the major coat protein from adenovirus type 2, has been refined at 2.9 A resolution. Hexon is a homo-trimer (molecular mass 3 x 109,077 Da) and crystallizes in the cubic space group P2(1)3, with a cell edge of 150.5 A. There are four molecules in the unit cell so that the crystallographic asymmetric unit contains one subunit of the trimer. The electron density in most regions is well-defined and 880 amino acid residues, of the 967 in this unusually long polypeptide chain, have been located and fitted. The N terminus (1 to 43) and three internal stretches (192 to 203, 270 to 291 and 444 to 453) are not defined, and a stretch (168 to 207) with unclear side-chain density is modelled as poly(Ala/Gly). The current refined model, consisting of 6943 non-hydrogen protein atoms and 85 water molecules, yields an R-factor of 19.9% for 18,176 reflections in the resolution range 5.0 to 2.9 A. The model has reasonable geometry with root-mean-square deviations from ideal bond lengths of 0.022 A and angle-related 1-3 distances of 0.056 A. The overall shape of the trimeric hexon molecule is unusual and may be divided into a pseudo-hexagonal base rich in beta-structure, and a triangular top formed from three long loops containing some secondary structure. The base contains two similar pedestal domains, P1 and P2, each of which is a flattened eight-stranded beta-barrel with the "jelly-roll greek key" topology characteristic of other viral coat proteins. P1 and P2 are related by an approximate 6-fold operation about the molecular 3-fold axis so that six barrels form the walls of the tubular hexon base. The hexon bases form close-packed p3 arrays on each facet of the icosahedral adenovirus virion. Unlike other viral capsids, the barrel axes are almost perpendicular to rather than parallel with the capsid surface. The hexon top, which consists of intimately interacting loops emerging from P1 and P2 in the base, has a triangular outline and so does not exhibit the pseudo-symmetry of the base. The structure of the hexon trimer shows how economically it meets the demands of its function as a stable protective viral coat, reveals the significance of the special features in its unusual amino acid sequence, and explains its biochemical and immunological properties. The molecule is hollow, with a large central cavity, and so has a high effective volume for its mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Magnetic circular dichroism spectroscopy as a probe of axial heme ligand replacement in semisynthetic mutants of cytochrome c.

Horse heart cytochrome c with either histidine or cysteine replacing the endogenous axial methionine ligand at position 80 has been characterized with magnetic circular dichroism (MCD) spectroscopy in the UV-visible region. Comparison of the MCD spectra of the mutant proteins in the ferric state to those of authentic bis-imidazole- and imidazole/thiolate-ligated ferric heme proteins clearly shows that the histidine-imidazole and cysteine-thiolate groups of the replacement amino acids at position 80 are coordinated to the heme iron in the mutant proteins. This study demonstrates the power of MCD spectroscopy in identifying axial ligands in mutant heme proteins. Accurate axial ligand assignment is essential for proper interpretation of the altered properties of such novel proteins.