Alasdair C. Steven

The Sm-like Hfq Protein Increases OxyS RNA Interaction with Target mRNAs

The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation. Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells. Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU. We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins.

The molecular biology of intermediate filaments

From the hairs on our head to the soles of our feet, our bodies are composed of cells rich in intracellular fibrous proteins called intermediate filaments (IF). Immunological and biochemical data established the existence of five subclasses of IF proteins that can form IF in our various tissues: a complex group of 30 or so keratins of 40-70 kd in epithelia; a single protein desmin of 52 kd in muscle; a single protein vimentin of 53 kd in cells of mesenchymal origin; glial fibrillary acidic protein (GFAP), a single protein of 50 kd in astroglia; and a triplet of neurofilament proteins, NF-L (about 65 kd), NF-M (about 105 kd), and NF-H (about 135 kd) in neuronal cells (Lazarides, 1980; 1982; Steinert, 1981; Zackroff et al., 1981; Steinert et al., 1984a; Weber and Geisler, 1984). The recent plethora of amino acid sequence information now allows a more rigorous classification of these proteins into three (or more) distinct sequence Types (see below). There is growing evidence that our invertebrate passengers and all of their relatives also contain most of these subclasses and sequence Types (Gilbert et al., 1975; Koury and Eckert, 1984; Walter and Biessmann, 1984). Having passed beyond an infancy of phenomenology, the field of IF has now matured to the point where it is being explored by the various approaches generically termed “molecular biology.” This work has largely been directed toward finding answers to fundamental questions about the structure, complexity, origins, expression, and functions of IF. At this time, there are no complete answers to any of these questions, although much progress has been made. In this short review, we examine the most recent data that impinge on these questions.

Identification of a major keratinocyte cell envelope protein, loricrin

During epidermal cell cornification, the deposition of a layer of covalently cross-linked protein on the cytoplasmic face of the plasma membrane forms the cell envelope. We have isolated and characterized cDNA clones encoding a major differentiation product of mouse epidermal cells, which has an amino acid composition similar to that of purified cell envelopes. Transcripts of this gene are restricted to the granular layer and are as abundant as the differentiation-specific keratins, K1 and K10. An antiserum against a C-terminal peptide localizes this protein in discrete granules in the stratum granulosum and subsequently at the periphery of stratum corneum cells. Immunofluorescence and immunoelectron microscopy detect this epitope only on the inner surface of purified cell envelopes. Taken together, these results suggest that it is a major component of cell envelopes. On the basis of its presumed function, this protein is named loricrin.

Influenza virus pleiomorphy characterized by cryoelectron tomography.

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles. In this study, we used cryoelectron tomography to visualize the 3D structures of 110 individual virions of the X-31 (H3N2) strain of influenza A. The tomograms distinguish two kinds of glycoprotein spikes [hemagglutinin (HA) and neuraminidase (NA)] in the viral envelope, resolve the matrix protein layer lining the envelope, and depict internal configurations of ribonucleoprotein (RNP) complexes. They also reveal the stems that link the glycoprotein ectodomains to the membrane and interactions among the glycoproteins, the matrix, and the RNPs that presumably control the budding of nascent virions from host cells. Five classes of virions, four spherical and one elongated, are distinguished by features of their matrix layer and RNP organization. Some virions have substantial gaps in their matrix layer (“molecular fontanels”), and others appear to lack a matrix layer entirely, suggesting the existence of an alternative budding pathway in which matrix protein is minimally involved.

Encapsidated Conformation of Bacteriophage T7 DNA

The structural organization of encapsidated T7 DNA was investigated by cryo-electron microscopy and image processing. A tail-deletion mutant was found to present two preferred views of phage heads: views along the axis through the capsid vertex where the connector protein resides and via which DNA is packaged; and side views perpendicular to this axis. The resulting images reveal striking patterns of concentric rings in axial views, and punctate arrays in side views. As corroborated by computer modeling, these data establish that the T7 chromosome is spooled around this axis in approximately six coaxial shells in a quasi-crystalline packing, possibly guided by the core complex on the inner surface of the connector.

Enzymatic and Structural Similarities between the Escherichia coli ATP-dependent Proteases, ClpXP and ClpAP*

Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (M r46,000) associate to form a six-membered ring (M r ∼ 280,000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (∼2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min−1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

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.

Outcome of the first electron microscopy validation task force meeting

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.

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.