Imaging has become an essential component in many jields of' medical and laboratory research and clinical practice. Biologists s t t i 4 cells and generate 3 0 confocal microscopy da fasets, virologists generate 3 0 reconstructions of' viruses jrom micrographs, radiologists idcnt{b cind qimntib tumors ,finm MRI ond CT scans, rind nenroscientists detect regioncrl metabolic brain activiy jrom PET andjirnctionnl MRI scans. Analysis ojthese diverse image types requires sophisticated computerized qiiantifica tion and visiializntion tools. Until recently, three-dimensional visircrlizrition o j images and quantitative analysis coiild only be perjbrmed using espensive UNIX workstations and ciistomized sojiware. Toda,v. much ofthe visualization and analysis can be petjbrmed on an inespensive desktop cornpiiter equipped with the appropriate graphics hardware und sojiware. This paper introduces an e.utensible plalfi,rmindependent, general-purpose image processing and visimlizotion program specifcal[v designed to meet the needs of'a Internet-linked medical research community. The application nnmed MIPA V (Medical Image Processing Analji.yis and Visiiolization) enables clinical and qrrantitative analysis of medical images over the Internet. Using MIPA V's standard tiserintetjface and analysis tools, researchers and clinicians at remote sites can easily share research data and analyses, thereby enhancing their ability to study. diagnose. monitor. and treat medical disorders.

No 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.Papillomaviruses are a family of structurally similar nonenveloped DNA viruses that infect stratified squamous epithelial tissues, such as the skin or mucous membranes, of humans and various animal species (reviewed in reference (17). A group of roughly 30 sexually transmitted human papillomavirus (HPV) types tend to infect the skin or the mucosal surfaces of the genitals. Although most genital HPV infections are clinically inapparent and self-limiting, persistent infection with oncogenic HPV types, such as HPV16, can lead to the development of cervical cancer or other anogenital cancers (reviewed in reference 47).The papillomavirus major capsid protein L1 can spontaneously self-assemble into 72-pentamer virus-like particles (VLPs) that closely resemble the native Tϭ7 icosahedral structure of papillomavirus virions (29). The structure of a truncated Tϭ1 12-pentamer L1 VLP has been solved at 3.5-Å resolution (9, 35). Naturally produced authentic papillomavirus virions also contain a minor capsid protein, L2 (20). Although L2 is dispensable for capsid formation, it has been implicated in encapsidation of the 8-kb circular doublestranded DNA viral genome (25, 54). L2 has also been shown to participate in multiple steps during papillomavirus entry into host cells. Proposed roles for L2 include the induction of conformational changes in cell-bound virions, disruption of endosomal membranes, and the subcellular t...

Ostreid herpesvirus 1 (OsHV-1) is the only member of the Herpesviridae that has an invertebrate host and is associated with sporadic mortality in the Pacific oyster (Crassostrea gigas) and other bivalve species. Cryo-electron microscopy of purified capsids revealed the distinctive T=16 icosahedral structure characteristic of herpesviruses, although the preparations examined lacked pentons. The gross genome organization of OsHV-1 was similar to that of certain mammalian herpesviruses (including herpes simplex virus and human cytomegalovirus), consisting of two invertible unique regions (U L , 167?8 kbp; U S , 3?4 kbp) each flanked by inverted repeats (TR L /IR L , 7?6 kbp; TR S /IR S , 9?8 kbp), with an additional unique sequence (X, 1?5 kbp) between IR L and IR S . Of the 124 unique genes predicted from the 207 439 bp genome sequence, 38 were members of 12 families of related genes and encoded products related to helicases, inhibitors of apoptosis, deoxyuridine triphosphatase and RING-finger proteins, in addition to membrane-associated proteins. Eight genes in three of the families appeared to be fragmented. Other genes that did not belong to the families were predicted to encode DNA polymerase, the two subunits of ribonucleotide reductase, a helicase, a primase, the ATPase subunit of terminase, a RecB-like protein, additional RING-like proteins, an ion channel and several other membrane-associated proteins. Sequence comparisons showed that OsHV-1 is at best tenuously related to the two classes of vertebrate herpesviruses (those associated with mammals, birds and reptiles, and those associated with bony fish and amphibians). OsHV-1 thus represents a third major class of the herpesviruses. INTRODUCTIONViruses are assigned to the family Herpesviridae on the basis of morphological criteria and have been identified in a wide range of vertebrates and one invertebrate, the Pacific oyster, Crassostrea gigas (Minson et al., 2000). The vertebrate herpesviruses fall into two major phylogenetic groups. Those in the first group have mammalian or avian hosts and are classified into three subfamilies (Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae) that share extensive genetic relationships . Reptilian herpesviruses probably also belong among the Alphaherpesvirinae (Nigro et al., 2004;Quackenbush et al., 1998;Une et al., 2000;Yu et al., 2001). Viruses in the second group infect amphibians or bony fish and again are interrelated (Bernard & Mercier, 1993;Davison, 1998; Davison et al., 1999). Genetic evidence for a common evolutionary origin for the two groups is tenuous, however, since not a single herpesvirus-specific gene is detectably conserved in both. The only completely sequenced lower vertebrate herpesvirus, channel catfish virus (CCV), does share a few genes with the higher vertebrate group, but these have counterparts in other organisms (Davison, 1992). The conserved gene that comes closest to being herpesvirus specific encodes the putative ATPase subunit of the terminase, an enzyme complex involved in p...

No abstract

Energy-dependent protein degradation is carried out by large multimeric protein complexes such as the proteasomes of eukaryotic and archaeal cells and the ATP-dependent proteases of eubacterial cells. Clp protease, a major multicomponent protease of Escherichia coli, consists of a proteolytic component, ClpP, in association with an ATP-hydrolyzing, chaperonin-like component, ClpA. To provide a structural basis for understanding the regulation and mechanism of action of Clp protease, we have used negative staining electron microscopy and image analysis to examine ClpA and ClpP separately, as well as active ClpAP complexes. Digitized images of ClpP and ClpA were analyzed using a novel algorithm designed to detect rotational symmetries. ClpP is composed of two rings of seven subunits superimposed in bipolar fashion along the axis of rotational symmetry. This structure is similar to that formed by the beta subunits of the eukaryotic and archaeal proteasomes. In the presence of MgATP, ClpA forms an oligomer with 6-fold symmetry when viewed en face. Side views of ClpA indicate that the subunits are bilobed with the respective domains forming two stacked rings. ClpAP complexes contain a tetradecamer of ClpP flanked at one or both ends with a hexamer of ClpA, resulting in a symmetry mismatch between the axially aligned molecules. Our findings demonstrate that, despite the lack of sequence similarity between ClpAP and proteasomes, these multimeric proteases nevertheless have a profound similarity in their underlying architecture that may reflect a common mechanism of action.

UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T = 16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent site that is not seen at other quasiequivalent sites or on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25 null capsids, and denaturant-extracted capsids, we conclude that (1) the C-capsid-specific component is a heterodimer of UL25 and UL17, and (2) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.

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 ϳ22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ϳ4% 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.Poliovirus, like related picornaviruses, is a small nonenveloped virus consisting of a plus-sense RNA genome enclosed within a protein shell or capsid (reviewed in reference 53). The capsid consists of 60 copies each of four proteins (VP1, VP2, VP3, and VP4) arranged on an icosahedral lattice. VP1, VP2, and VP3 have similar wedge-shaped cores, each an eightstranded beta-barrel (the strands are designated B, I, D, G, C, H, E, and F), but each protein has unique loops connecting the strands and unique N and C termini (31). VP4 is small, myristylated (15), and has an extended structure.The wedge-shaped cores of the subunits form the closed protein shell of the virion, with five copies of VP1 packing around the fivefold axes and VP2 and VP3 alternating around the threefold axes of the particle. The virion is stabilized by interactions among the wedge-shaped cores and by a network-on the inner surface of the protein shell-that is formed by VP4 and the N-terminal extensions of VP1, VP2, and VP3. The high-resolution structure of an empty capsid assembly intermediate shows that formation of the internal network is dependent on a late proteolytic cleavage of the capsid protein precursor, VP0, to yield VP4 and VP2 (5). This "maturation cleavage" is associated with the encapsidation of the viral RNA and is required for virion stability. Unfortunately, the viral RNA (which lacks the icosahedral symmetry of the protein coat) is not visible in the high-resolution structure of the virus, and its role in stabilizing the virus structure is unknown.Despite extensive chemical and molecular characterization, the mechanism of picornavirus cell entry is known only in outline (14,48,49,(51)(52)(53)59). In order to initiate a productive infection, the viral RNA must be externalized, cross a membrane, and be delivered to the cytoplasm. Infection begins with binding of the virion to a receptor that is a member of the immunoglobulin superfamily (37, 45), whereup...