Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage phi29.

A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages. Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry. Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component. This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of phi29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch. A reconstruction of phi29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the phi29 DNA packaging motor.

Complete in vitro assembly of the reovirus outer capsid produces highly infectious particles suitable for genetic studies of the receptor-binding protein.

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins--sigma1, mu1, and sigma3--to enter host cells. sigma1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma1, mu1, and sigma3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma1. The recoated particles bound to and infected cultured cells in a sigma1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma1 protein. Additional experiments showed that recoated particles containing sigma1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma1 with respect to its structure, assembly into particles, and roles in entry.

The structure of isometric capsids of bacteriophage T4.

The three-dimensional structure of DNA-filled, bacteriophage T4 isometric capsids has been determined by means of cryoelectron microscopy and image reconstruction techniques. The packing geometry of protein subunits on the capsid surface was confirmed to be that of the triangulation class T = 13. The reconstruction clearly shows pentamers, attributed to capsid protein gp24*, surrounded by hexamers of the major capsid protein, gp23*. Positions of the accessory proteins, Hoc and Soc, are also clearly delineated in the surface lattice. The Hoc protein is the most prominent surface feature and appears as an extended molecule with a rounded base from which a thin neck and a globular head protrude. One Hoc molecule associates with each hexamer. Nearly continuous "ridges" are formed at the periphery of the gp23* hexamers by an association of 12 Soc molecules; however, Soc is absent along the boundaries between the hexamers and the pentamers. The duplex DNA genome forms a highly condensed series of concentric layers, spaced about 2.36 nm apart, that follow the general contour of the inner wall of the protein capsid.

Structure of the Maize streak virus geminate particle.

The Geminiviridae is an extensive family of plant viruses responsible for economically devastating diseases in crops worldwide. Geminiviruses package circular, single-stranded DNA (ssDNA) genomes. The characteristic twinned or "geminate" particles, which consist of two joined, incomplete T = 1 icosahedra, are unique among viruses. We have determined the first structure of a geminivirus particle, the Nigerian strain of Maize streak virus (MSV-N), using cryo-electron microscopy and three-dimensional image reconstruction methods. The particle, of dimensions 220 x 380 A, has an overall 52-point-group symmetry, in which each half particle "head" consists of the coat protein (CP) arranged with quasi-icosahedral symmetry. We have modeled the MSV-N CP as an eight-stranded, antiparallel beta-barrel motif (a structural motif common to all known ssDNA viruses) with an N-terminal alpha-helix. This has produced a model of the geminate particle in which 110 copies of the CP nicely fit into the reconstructed density map. The reconstructed density map and MSV-N pseudo-atomic model demonstrate that the geminate particle has a stable, defined structure.

The organization of mature Rous sarcoma virus as studied by cryoelectron microscopy.

We have studied the organization of mature infectious Rous sarcoma virus (RSV), suspended in vitreous ice, using transmission electron microscopy. The enveloped virions are spherical in shape, have a mean diameter of 127 nm, and vary significantly in size. Image processing reveals the presence of the viral matrix protein underlying the lipid bilayer and the viral envelope proteins external to the lipid bilayer. In the interior of the virus, the characteristic mature retroviral core is clearly imaged. In contrast to lentiviruses, such as human immunodeficiency virus, the core of RSV is essentially isometric. The capsid, or external shell of the core, has a faceted, almost polygonal appearance in electron micrographs, but many capsids also exhibit continuous surface curvature. Cores are not uniform in size or shape. Serrations observed along the projected faces of the core suggest a repetitive molecular structure. Some isolated cores were observed in the sample, confirming that cores are at least transiently stable in the absence of the viral envelope. Using an approach grounded in geometric probability, we estimate the size of the viral core from the projection data. We show that the size of the core is not tightly controlled and that core size and virion size are positively correlated. From estimates of RNA packing density we conclude that either the RNA within the core is loosely packed or, more probably, that it does not fill the core.

Structure of the bacteriophage phi29 DNA packaging motor.

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate double-stranded DNA into preformed capsids (proheads) of bacterial viruses and certain animal viruses. Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage phi29 into a precursor capsid. We determined the structure of the head-tail connector--the central component of the phi29 DNA packaging motor--to 3.2 A resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNA-ATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.

Babesiosis in a renal transplant recipient acquired through blood transfusion.

BACKGROUND: The success of organ-replacement therapies has resulted in a population of chronically immunosuppressed but active people who experience increased vulnerability to tick-borne zoonoses. Several of these infections may be life threatening. Human babesiosis is an emerging zoonosis that is transmitted by the same tick that transmits Lyme disease and human granulocytic ehrlichiosis. METHODS: We briefly review these zoonoses and present a case of a renal transplant recipient who survived infection by Babesia microti contracted through blood transfusion. RESULTS: A recipient of a living-related renal transplant developed acute postoperative hemolytic anemia. The etiology of this anemia was diagnosed by peripheral red blood cell smear as Babesia microti. The patient was managed by a reduction in transplant immunosuppressive therapy and administration of clindamycin and quinine antimicrobials. CONCLUSIONS: Transplant patients may contract babesiosis after tick exposure and/or via blood transfusion. The diagnosis of babesiosis may be confused with malaria and should be included in the differential diagnosis of posttransplant hemolytic-uremic syndrome in organ transplant patients.

Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs.

Viruses are cellular parasites. The linkage between viral and host functions makes the study of a viral life cycle an important key to cellular functions. A deeper understanding of many aspects of viral life cycles has emerged from coordinated molecular and structural studies carried out with a wide range of viral pathogens. Structural studies of viruses by means of cryo-electron microscopy and three-dimensional image reconstruction methods have grown explosively in the last decade. Here we review the use of cryo-electron microscopy for the determination of the structures of a number of icosahedral viruses. These studies span more than 20 virus families. Representative examples illustrate the use of moderate- to low-resolution (7- to 35-A) structural analyses to illuminate functional aspects of viral life cycles including host recognition, viral attachment, entry, genome release, viral transcription, translation, proassembly, maturation, release, and transmission, as well as mechanisms of host defense. The success of cryo-electron microscopy in combination with three-dimensional image reconstruction for icosahedral viruses provides a firm foundation for future explorations of more-complex viral pathogens, including the vast number that are nonspherical or nonsymmetrical.

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor.

Two human rhinovirus serotypes complexed with two- and five-domain soluble fragments of the cellular receptor, intercellular adhesion molecule-1, have been investigated by X-ray crystallographic analyses of the individual components and by cryo-electron microscopy of the complexes. The three-dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N-terminal domain of the receptor binds to the rhinovirus 'canyon' surrounding the icosahedral 5-fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N-terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.

RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein.

Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. The results presented here reveal a striking in vivo switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.

Three-dimensional structure of Aleutian mink disease parvovirus: implications for disease pathogenicity.

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.

Mechanism of zeolite A nanocrystal growth from colloids at room temperature.

The formation and growth of crystal nuclei of zeolite A from clear solutions at room temperature were studied with low-dose, high-resolution transmission electron microscopy in field emission mode and with in situ dynamic light scattering. Single zeolite A crystals nucleated in amorphous gel particles of 40 to 80 nanometers within 3 days at room temperature. The resulting nanoscale single crystals (10 to 30 nanometers) were embedded in the amorphous gel particles. The gel particles were consumed during further crystal growth at room temperature, forming a colloidal suspension of zeolite A nanocrystals of 40 to 80 nanometers. On heating this suspension at 80 degrees C, solution-mediated transport resulted in additional substantial crystal growth.

Assembly of a tailed bacterial virus and its genome release studied in three dimensions.

We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryoelectron microscopy and X-ray crystallography of virus-Fab complexes.

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the beta-B-beta-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.

Comparison of the native CCMV virion with in vitro assembled CCMV virions by cryoelectron microscopy and image reconstruction.

Cryoelectron microscopy and three-dimensional image reconstruction analysis has been used to determine the structure of native and in vitro assembled cowpea chlorotic mottle virus (CCMV) virions and capsids to 25-A resolution. Purified CCMV coat protein was used in conjunction with in vitro transcribed viral RNAs to assemble RNA 1 only, RNA 2 only, RNA 3/4 only, and empty (RNA lacking) virions. The image reconstructions demonstrate that the in vitro assembled CCMV virions are morphologically indistinguishable from native virions purified from infected plants. The viral RNA (vRNA) is packaged similarly within the different types of virions. The centers of all assembled particles are generally devoid of density and the vRNA packs against the interior surface of the virion shell. The vRNA appears to adopt an ordered conformation at each of the quasi-threefold axes.

A method for establishing the handedness of biological macromolecules.

When biological macromolecules are imaged in the transmission electron microscope (TEM), their inherent handedness is lost because the three-dimensional (3D) structure is projected onto a two-dimensional (2D) plane, and identical 2D projections can be made from either 3D enantiomer. Nevertheless, tilt experiments in the TEM can be used to determine handedness. These experiments have been performed successfully on negatively stained specimens. More recently, the method was applied to unstained, frozen-hydrated specimens imaged by means of cryoelectron microscopy (cryoTEM) methods. Tilt experiments involve recording two micrographs of the same particles at different tilt angles, computing enantiomeric reconstructions from particle images in one micrograph, predicting orientations of corresponding particles in the second micrograph, and comparing model projections with particle images in the second micrograph. In principle, this procedure can be used to determine the handedness of any biological macromolecule imaged by cryoTEM, provided the enantiomeric reconstructions are distinguishable.

Localization of a C-terminal region of lambda2 protein in reovirus cores.

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.

Neutralizing antibody to human rhinovirus 14 penetrates the receptor-binding canyon.

The three-dimensional structure of intact human rhinovirus 14 (HRV-14) complexed with Fab fragments (Fab17-IA) from a strongly neutralizing antibody that binds bivalently to the virion has been determined to 4.0 angstrom resolution by a combination of X-ray crystallography and cryo-electron microscopy. In contradiction to the most commonly held model of antibody-mediated neutralization, Fab17-IA does not induce a conformational change in the HRV-14 capsid. Instead, the paratope of the antibody undergoes a large conformational change to accommodate the epitope. Unlike any previously described antibody-antigen structure, the conserved framework region of the antibody makes extensive contact with the viral surface. Fab17-IA penetrates deep within the canyon in which the cellular receptor for HRV-14 binds. Hence, it is unlikely that viral quaternary structure evolves merely to evade immune recognition. Instead, the shape and position of the receptor-binding region on a virus probably dictates receptor binding and subsequent uncoating events and has little or no influence on concealing the virus from the immune system.

Conserved features in papillomavirus and polyomavirus capsids.

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.