Benes L. Trus

Medical Image Processing, Analysis and Visualization in clinical research

Imaging has become an essential component in many fields of medical and laboratory research and clinical practice. Biologists study cells and generate 3D confocal microscopy data sets; virologists generate 3D reconstructions of viruses from micrographs; radiologists identify and quantify tumors from MRI and CT scans; and neuroscientists detect regional metabolic brain activity from PET and functional MRI scans. Analysis of these diverse image types requires sophisticated computerized quantification and visualization tools. Until recently, 3D visualization of images and quantitative analysis could only be performed using expensive UNIX workstations and customized software. Today, much of the visualization and analysis can be performed on an inexpensive desktop computer equipped with the appropriate graphics hardware and software. This paper introduces an extensible, platform-independent, general-purpose image processing and visualization program specifically designed to meet the needs of an Internet-linked medical research community. The application, named MIPAV (Medical Image Processing, Analysis and Visualization), enables clinical and quantitative analysis of medical images over the Internet. Using MIPAVs standard user interface and analysis tools, researcher and clinicians at remote sites can easily share research data and analyses, thereby enhancing their ability to study, diagnose, monitor and treat medical disorders.

Atomic model of the papillomavirus capsid

Papillomaviruses propagate in differentiating skin cells, and certain types are responsible for the onset of cervical cancer. We have combined image reconstructions from electron cryomicroscopy (cryoEM) of bovine papillomavirus at 9 Å resolution with coordinates from the crystal structure of small virus‐like particles of the human papillomavirus type 16 L1 protein to generate an atomic model of the virion. The overall fit of the L1 model into the cryoEM map is excellent, but residues 402–446 in the ‘C‐terminal arm’ must be rebuilt. We propose a detailed model for the structure of this arm, based on two constraints: the presence of an intermolecular disulfide bond linking residues 175 and 428, and the clear identification of a feature in the image reconstruction corresponding to an α‐helix near the C‐terminus of L1. We have confirmed the presence of the disulfide bond by mass spectrometry. Our ‘invading arm’ model shows that papilloma‐ and polyomaviruses have a conserved capsid architecture. Most of the rebuilt C‐terminal arm is exposed on the viral surface; it is likely to have a role in infection and in immunogenicity.

Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus

The organization of DNA within the HSV-1 capsid has been determined by cryoelectron microscopy and image reconstruction. Purified C-capsids, which are fully packaged, were compared with A-capsids, which are empty. Unlike A-capsids, C-capsids show fine striations and punctate arrays with a spacing of approximately 2.6 nm. The packaged DNA forms a uniformly dense ball, extending radially as far as the inner surface of the icosahedral (T = 16) capsid shell, whose structure is essentially identical in A-capsids and C-capsids. Thus we find no evidence for the inner T = 4 shell previously reported by Schrag et al. to be present in C-capsids. Encapsidated HSV-1 DNA closely resembles that previously visualized in bacteriophages T4 and lambda, thus supporting the idea of a close parallelism between the respective assembly pathways of a major family of animal viruses (the herpesviruses) and a major family of bacterial viruses.

Arrangement of L2 within the Papillomavirus Capsid

ABSTRACT Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein, L2, is not required for capsid formation, it is thought to participate in encapsidation of the viral genome and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. To address these questions, we developed methods for serial propagation of infectious HPV16 capsids (pseudoviruses) in cultured human cell lines. Biochemical analysis of capsid preparations produced using various methods showed that up to 72 molecules of L2 can be incorporated per capsid. Cryoelectron microscopy and image reconstruction analysis of purified capsids revealed an icosahedrally ordered L2-specific density beneath the axial lumen of each L1 capsomer. The relatively close proximity of these L2 density buttons to one another raised the possibility of homotypic L2 interactions within assembled virions. The concept that the N and C termini of neighboring L2 molecules can be closely apposed within the capsid was supported using bimolecular fluorescence complementation or “split GFP” technology. This structural information should facilitate investigation of L2 function during the assembly and entry phases of the papillomavirus life cycle.

Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus

ABSTRACT Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ∼22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ∼4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 Å resolution

The three-dimensional structure of bovine papillomavirus has been determined to 9 Å resolution by reconstruction of high resolution, low dose cryo-electron micrographs of quench-f rozen virions. Although hexavalent and pentavalent capsomeres form star-shaped pentamers of the major capsid protein L1, they have distinct high-resolution structures. Most prominently, a 25 Å hole in the centre of hexavalent capsomeres is occluded in the pentavalent capsomeres. This raises the possibility that the L2 minor capsid protein is located in the centre of the pentavalent capsomeres. Inter-capsomere connections ∼10 Å in diameter were clearly resolved. These link adjacent capsomeres and are reminiscent of the helical connections that stabilize polyomavirus.

Molecular substructure of a viral receptor-recognition protein. The gp17 tail-fiber of bacteriophage T7.

The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400). We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes. This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained. Mass measurements made from the latter images establish that the fiber is a trimer of gp17. The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151. The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis. The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube. We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.

Structure and Polymorphism of the UL6 Portal Protein of Herpes Simplex Virus Type 1

ABSTRACT By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry. The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 Å, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site. Its architecture resembles those of bacteriophage portal/connector proteins.

Isolation of Herpes Simplex Virus Procapsids from Cells Infected with a Protease-Deficient Mutant Virus

ABSTRACT Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids—normally, short-lived intermediates—would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0°C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-Å resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated withm100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.

Capsid Structure of Kaposi's Sarcoma-Associated Herpesvirus, a Gammaherpesvirus, Compared to Those of an Alphaherpesvirus, Herpes Simplex Virus Type 1, and a Betaherpesvirus, Cytomegalovirus

ABSTRACT The capsid of Kaposis sarcoma-associated herpesvirus (KSHV) was visualized at 24-Å resolution by cryoelectron microscopy. Despite limited sequence similarity between corresponding capsid proteins, KSHV has the same T=16 triangulation number and much the same capsid architecture as herpes simplex virus (HSV) and cytomegalovirus (CMV). Its capsomers are hexamers and pentamers of the major capsid protein, forming a shell with a flat, close-packed, inner surface (the “floor”) and chimney-like external protrusions. Overlying the floor at trigonal positions are (αβ2) heterotrimers called triplexes. The floor structure is well conserved over all three viruses, and the most variable capsid features reside on the outer surface, i.e., in the shapes of the protrusions and triplexes, in which KSHV resembles CMV and differs from HSV. Major capsid protein sequences from the three subfamilies have some similarity, which is closer between KSHV and CMV than between either virus and HSV. The triplex proteins are less highly conserved, but sequence analysis identifies relatively conserved tracts. In alphaherpesviruses, the α-subunit (VP19c in HSV) has a 100-residue N-terminal extension and an insertion near the C terminus. The small basic capsid protein sequences are highly divergent: whereas the HSV and CMV proteins bind only to hexons, difference mapping suggests that the KSHV protein, ORF65, binds around the tips of both hexons and pentons.