Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once "uncoated" in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 degrees C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 degrees C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus.

The complete nucleotide sequences of two double-stranded (ds) RNA molecules, S1 (1,744 bp) and S2 (1,567 bp), isolated from an isolate HP62 of the Himalayan Dutch elm disease fungus, Ophiostoma himal-ulmi, were determined. RNA S1 had the potential to encode a protein, P1, of 539 amino acids (62.7 kDa), which contained sequence motifs characteristic of RNA-dependent RNA polymerases (RdRps). A database search showed that P1 was closely related to RdRps of members of the genus Partitivirus in the family Partitiviridae. RNA S2 had the potential to encode a protein, P2, of 430 amino acids (46.3 kDa), which was related to capsid proteins of members of the genus Partitivirus. Virus particles isolated from isolate HP62 were shown to be isometric with a diameter of 30 nm, and to contain dsRNAs S1 and S2 and a single capsid protein of 46 kDa. N-terminal sequencing of tryptic peptides derived from the capsid protein proved unequivocally that it is encoded by RNA S2 and corresponds to protein P2. It is concluded that O. himal-ulmi isolate HP62 contains a new member of the genus Partitivirus, which is designated Ophiostoma partitivirus 1. A phylogenetic tree of RdRps of members of the family Partitiviridae showed that there are least two RdRp lineages of viruses currently classified in the genus Partitivirus. One of these lineages contained viruses with fungal hosts and viruses with plant hosts, raising the possibility of horizontal transmission of partitiviruses between plants and fungi. The partitivirus RdRp and capsid proteins appear to have evolved in parallel with the capsid proteins evolving much faster than the RdRps.

Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are translocated, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex.

Gram-negative bacteria commonly interact with eukaryotic host cells using type III secretion systems (TTSSs or secretons), which comprise cytoplasmic, transmembrane and extracellular domains. The extracellular domain is a hollow needle-like structure protruding 60nm beyond the bacterial surface. The TTSS is activated to transfer bacterial proteins directly into a host cell only upon physical contact with the target cell. We showed previously that the monomer of the Shigella flexneri needle, MxiH, assembles into a helical structure with parameters similar to those defining the architecture of the extracellular components of bacterial flagella. By analogy with flagella, which are known to exist in different helical states, we proposed that changes in the helical packing of the needle might be used to sense host cell contact. Here, we show that, on the contrary, mutations within MxiH that lock the TTSS into altered secretion states do not detectably alter the helical packing of needles. This implies that either: (1) host cell contact is signalled through the TTSS via helical changes in the needle that are significantly smaller than those linked to structural changes in the flagellar filament and therefore too small to be detected by our analysis methods or (2) that signal transduction in this system occurs via a novel molecular mechanism.

Voltage-sensitive K(+) channels (Kv) serve numerous important roles, e.g. in the control of neuron excitability and the patterns of synaptic activity. Here, we use electron microscopy (EM) and single particle analysis to obtain the first, complete structure of Kv1 channels, purified from rat brain, which contain four transmembrane channel-forming alpha-subunits and four cytoplasmically-associated beta-subunits. The 18A resolution structure reveals an asymmetric, dumb-bell-shaped complex with 4-fold symmetry, a length of 140A and variable width. By fitting published X-ray data for recombinant components to our EM map, the modulatory (beta)(4) was assigned to the innermost 105A end, the N-terminal (T1)(4) domain of the alpha-subunit to the central 50A moiety and the pore-containing portion to the 125A membrane part. At this resolution, the selectivity filter could not be localised. Direct contact of the membrane component with the central (T1)(4) domain occurs only via peripheral connectors, permitting communication between the channel and beta-subunits for coupling of responses to changes in excitability and metabolic status of neurons.

ABSTRACT BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.

ABSTRACT Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella, is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.

The mechanism for assembly of membrane lipid bilayers in vivo is generally considered to occur on existing membranes which act as templates for the assembly process. Not presently understood is the source of the membrane template. A thermodynamic theory describing a spontaneous process for assembly of unilamellar vesicles with properties of a critical state (1,2) has been tested in this study. The spontaneous formation of large unilamellar vesicles (LUV’s) from phospholipid components of cell membranes at a critical temperature T* has now been documented by cryo-electron microscope studies using aqueous dispersions of dimyrystoylphosphatidylcholine (T* = 29°C), and the total lipid extracts of E. coli membranes cultured at 32°C (T* = 32°C). The lipids were incubated at temperatures below, at and above the critical temperatures for LUV assembly. in conformity with thermodynamic theory, and other physical measurements that indicate unique bilayer properties exist at T* (3-5), LUV’s are seen to form only at T*.

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.

ABSTRACT An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37°C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to ∼30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

Newcomb and coworkers (W. W. Newcomb, F. L. Homa, D. R. Thomsen, F. P. Booy, B. L. Trus, A. C. Steven, J. V. Spencer, and J. C. Brown, J. Mol. Biol. 263:432–446, 1996; W. W. Newcomb, F. L. Homa, D. R. Thomsen, Z. Ye, and J. C. Brown, J. Virol. 68:6059–6063, 1994) have recently described an in vitro herpes simplex virus (HSV) capsid assembly product which, because of certain parallels between its properties and those of bacteriophage proheads, they have designated the procapsid. As in their bacteriophage counterparts, there are marked differences between the structures of the two types of particle, and conversion from the procapsid to the capsid form requires extensive reconfiguration of the subunits. This reconfiguration occurs spontaneously upon extended in vitro incubation. One of the distinctive features of the HSV procapsids is that, unlike mature capsids, they are unstable and disassemble upon storage at 2°C. Using a mutant of HSV type 1 (ts1201), which has a lesion in the protease responsible for maturational cleavage of the scaffolding protein, we have demonstrated that capsids present within cells infected at nonpermissive temperatures are also cryosensitive and disappear if the cells are incubated at 0°C. This suggests that ts1201 capsids may resemble procapsids in structure. However, ts1201 capsids remain cryosensitive following extended incubation at an elevated temperature and, therefore, do not appear to undergo the spontaneous reconfiguration seen with in vitro-assembled procapsids. The lesion in ts1201 is reversible, and capsids formed at the nonpermissive temperature can undergo maturational cleavage and go on to form infectious virions following downshift to permissive temperatures. The sensitivity of ts1201 capsids to low temperatures is closely correlated with the cleavage status of the scaffolding protein, suggesting that proteolysis may act to trigger their conversion to the stable form. The experiments described here provide the firmest evidence yet that the procapsid has a biologically relevant role in the virus life cycle.

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).

ATP-dependent proteases play a major role in regulatory protein degradation in both prokaryotic and eukaryotic cells. ATP-dependent proteases in E. coli fall into two classes. The first class requires the interaction of structurally separate proteases with an ATPase, whereas in the second class both the protease and ATPase are formed from regions of the same polypeptide chain. We have studied the structure of several of these protein degrading complexes in E. coli and have found a remarkable similarity in the architecture of these macromolecular assemblies. The prototypical protease of the first class has as its proteolytic component ClpP, a 14 subunit (MW 21,500) complex arranged as two lOnm-diameter stacked rings of seven subunits each. ClpP can interact with either one of two ATPases, ClpA or ClpX, each with unique substrate specificity. ClpA has two ATP-binding sites per subunit (MW 84,000), and its subunits are arranged as a 13nm (diameter) hexameric ring (MW -500,000).

Complexes between bovine papillomavirus type 1 (BPV1) and examples of two sets of neutralizing, monoclonal antibodies (mAb) to the major capsid protein (L1) were analyzed by low-dose cryo-electron microscopy and three-dimensional (3D) image reconstruction to 13 Å resolution. mAb #9 is representative of a set of neutralizing antibodies that can inhibit viral binding to the cell surface, while mAb 5B6 is representative of a second set that efficiently neutralizes papillomaviruses without significantly inhibiting viral binding to the cell surface. The 3D reconstructions reveal that mAb #9 binds to L1 molecules of both pentavalent and hexavalent capsomeres. In contrast, 5B6 binds only to hexavalent capsomeres, reflecting the significant structural or environmental differences for the 5B6 epitope in the 12 pentavalent capsomeres. Epitope localization shows that mAb #9 binds monovalently to the tips of capsomeres whereas 5B6 binds both monovalently and bivalently to the sides of hexavalent capsomeres approximately two-thirds of the way down from the outer tips, very close to the putative stabilizing intercapsomere connections. The absence of mAb 5B6 from the pentavalent capsomeres and its inability to prevent viral binding to the cell surface suggest that receptor binding may occur at one or more of the 12 virion vertices.

Individual dendrimer molecules of poly(amidoamine) (PAMAM) from generation 10 (G10) to G5 were imaged by conventional transmission electron microscopy (TEM) after staining with aqueous sodium phosphotungstate. The dendrimers were resolved as separate, beam-stable entities, and their sizes and distribution of sizes were statistically analyzed and compared with data from small-angle X-ray scattering in solution. Aqueous G10 dendrimers were also characterized by examination in the frozen, hydrated state after quench-freezing in liquid ethane (cryo-TEM) and the data compared with the results of the staining experiments. To a first approximation, the dendrimers appeared circular in projection and the diameters conformed to a Gaussian distribution which broadened somewhat with increasing generation number. Cryo-TEM, in general, confirmed the staining experiments but suggested that, in the native state, there is more variability in the shapes of the dendrimers, with polyhedral shapes occurring quite frequently.