Complex Membrane Remodeling during Virion Assembly of the 30,000-Year-Old Mollivirus Sibericum.

Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly, and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary three-dimensional (3D)-electron microscopy techniques, including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant, and DNA is packed within virions prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening, and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggest that the molecular mechanisms for cellular membrane remodeling and persistence are unique.IMPORTANCE Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the world and appear to be unique in several aspects. They belong to at least four viral families, and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of "Molliviridae," which was isolated from a 30,000-year-old permafrost sample and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna, (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled, and (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding of how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.

Mimivirus.

Acanthamoeba polyphaga Mimivirus, the first representative and prototype member of the Mimiviridae, is the latest addition to the menagerie of lesser-known big DNA viruses. Due to the size of its particle--a fiber-covered icosahedral protein capsid with a diameter of 0.7 microm--Mimivirus was initially mistaken for an intracellular parasitic bacteria. Its 1.2-Mb genome sequence was then found to encode more than 900 proteins, many of them associated with functions never before encountered in a virus, such as four aminoacyl-tRNA synthetases. The finding of Mimivirus-encoded central components of the protein translation apparatus thought to be the signature of cellular organisms revived the debate about the origin of DNA viruses and their possible role in the emergence of the eukaryotic cell. Despite the many features making it unique in the viral world, Mimivirus is nevertheless phylogenetically close to other large DNA viruses, such as phycodnaviruses and iridoviruses, and most likely share a common ancestry with all nucleocytoplasmic large DNA viruses. Postgenomic studies have now started in various laboratories, slowly shedding some light on the physiology of the largest and most complex virus isolated to date. This chapter summarizes our present knowledge on Mimivirus.

Annotation of bacterial genomes using improved phylogenomic profiles.

MOTIVATION: Phylogenomic profiling is a large-scale comparative genomic method used to infer protein function from evolutionary information first described in a binary form by Pellegrini et al. (1999). Here, we propose improvements of this approach including the use of normalized Blastp bit scores, a normalization of the matrix of profiles to take into account the evolutionary distances between bacteria, the definition of a phylogenomic neighborhood based on continuous pairwise distances between genes and an original annotation procedure including the computation of a p-value for each functional assignment. RESULTS: The method presented here increases the number of Ecocyc enzymes identified as being evolutionarily related by about 25% with respect to the original binary form (absent/present) method. The fraction of 'false' positives is shown to be smaller than 20%. Based on their phylogenomic relationships, genes of unknown function can then be automatically related to annotated genes. Each gene annotation predicted is associated with a p-value, i.e. its probability to be obtained by chance. The validity of this method was extensively tested on a large set of genes of known function using the MultiFun database. We find that 50% of 3122 function attributions that can be made at a p-value level of 10(-11) correspond to the actual gene annotation. The method can be readily applied to any newly sequenced microbial genome. In contrast to earlier work on the same topic, our approach avoids the use of arbitrary cut-off values, and provides a reliability estimate of the functional predictions in form of p-values.

Recent advances in computational genomics.

In the post-genomic era, the new discipline of functional genomics is now facing the challenge of associating a function (as well as estimating its relevance to industrial applications) to about 100,000 microbial, plant or animal genes of known sequence but unknown function. Besides the design of databases, computational methods are increasingly becoming intimately linked with the various experimental approaches. Consequently, bioinformatics is rapidly evolving into independent fields addressing the specific problems of interpreting i) genomic sequences, ii) protein sequences and 3D-structures, as well as iii) transcriptome and macromolecular interaction data. It is thus increasingly difficult for the biologist to choose the computational approaches that perform best in these various areas. This paper attempts to review the most useful developments of the last 2 years.

DNA gyrase-mediated natural resistance to fluoroquinolones in Ehrlichia spp.

Fluoroquinolone susceptibility heterogeneity between various Ehrlichia species has been previously demonstrated. In gram-negative bacteria, resistance to fluoroquinolones most often corresponds to specific amino acid variations in a portion of the protein sequence of the A subunit of DNA gyrase (GyrA), referred to as the quinolone resistance-determining region (QRDR). We suspected a similar mechanism to be responsible for natural resistance in some Ehrlichia species. To verify this hypothesis, we sequenced the entire gyrA gene of the quinolone-susceptible species Ehrlichia sennetsu and designed specific primers to amplify and sequence the QRDR of four other Ehrlichia species as well as the closely related species Cowdria ruminantium. We identified in the fluoroquinolone-resistant species Ehrlichia chaffeensis and Ehrlichia canis a specific GyrA QRDR amino acid sequence, also present in C. ruminantium (whose susceptibility to fluoroquinolones remains unknown). These three species belong to a single phylogenetic cluster referred to as the E. canis genogroup. A different GyrA QRDR pattern, shared by the Ehrlichia species representatives of the E. sennetsu and Ehrlichia phagocytophila genogroups, was identified. Three of the four species tested are known to be susceptible to fluoroquinolones. A serine residue in position 83 (Escherichia coli numbering) in the susceptible species is replaced by an alanine residue in fluoroquinolone-resistant species. These results are consistent with the current knowledge on fluoroquinolone resistance in other gram-negative bacteria. They are indicative of a natural gyrase-mediated resistance to fluoroquinolones in the E. canis genogroup.

Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme.

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression of Escherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K(i) approximately 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor of vertebrate lysozyme) at the E. coli genetic stock center.

Crystallization and preliminary crystallographic study of the peptidoglycan-associated lipoprotein from Escherichia coli.

The peptidoglycan-associated lipoprotein (Pal) from Escherichia coli is part of the Tol--Pal multiprotein complex used by group A colicins to penetrate and kill cells. Pal homologues are found in many Gram-negative bacteria and the Tol--Pal system is thought to play a role in bacterial envelope integrity. The Pal protein comprises 152 amino acids. Crystals of the C-terminal 109-amino-acid fragment of the Pal protein have been produced. The crystals belong to the tetragonal space group I4(1), with unit-cell parameters a = b = 89.3, c = 67.2 A. There are two molecules in the asymmetric unit. Frozen crystals diffract to at least 2.8 A resolution using synchrotron radiation. Selenomethionine-substituted truncated Pal protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing.

Crystallization and preliminary crystallographic study of the periplasmic domain of the Escherichia coli TolR protein.

The TolR protein from Escherichia coli is part of the Tol-Pal multiprotein complex used by group A colicins to penetrate and kill cells. All genes of the Tol-Pal system are conserved in Gram-negative bacteria and this system is thought to play a role in the maintenance of the bacterial envelope integrity, although its exact function is not known. The TolR protein comprises 142 amino acids. The periplasmic domain of the TolR protein has been expressed, purified and crystallized. The crystals belong to the tetragonal space group P4(1)22, with unit-cell parameters a = 46.3, c = 178.0 A. There are one or two molecules in the asymmetric unit. Frozen crystals diffract to at least 3.2 A resolution using synchrotron radiation. Selenomethionine-substituted periplasmic TolR protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing.

Crystallization and preliminary crystallographic study of b0220, an 'ORFan' protein of unknown function from Escherichia coli.

Newly sequenced microbial genomes continue to reveal up to 50% functionally uncharacterized 'anonymous' genes. A significant fraction of these anonymous ORFs does not exhibit any sequence similarity to any protein in the databases and constitutes a set of unique sequences, denoted 'ORFans'. The structure determination of ORFan proteins is both of evolutionary and functional interest. Here, the first crystallization of an Escherichia coli ORFan gene product, the 157 amino-acid b0220 protein, is reported. The crystals belong to the trigonal space group P3 or P3(1), with unit-cell parameters a = b = 47.2, c = 88.4 A. There are two molecules in the asymetric unit. Frozen crystals diffract to 1.6 A resolution using synchrotron radiation. Phasing was performed using multiwavelength anomalous dispersion (MAD) on the selenomethionine-substituted b0220 protein.

Crystallization and preliminary crystallographic study of an extremophile cytochrome c4 from Thiobacillus ferrooxidans.

Soluble periplasmic dihaemic cytochrome c(4), of 21 293 Da molecular mass, has been characterized from Thiobacillus ferrooxidans, an acidophilic bacteria. The native cytochrome has been purified from the bacteria using ion-exchange chromatography and crystallized using solution 27 of the Hampton Research Crystal Screen II. The crystals belong to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = 101.59, b = 101.59, c = 151.59 A. Frozen crystals diffract to 2.17 A resolution. The MAD method is currently being used (four Fe atoms per asymmetric unit) to solve the protein structure.

Structure of the Escherichia coli TolB protein determined by MAD methods at 1.95 A resolution.

BACKGROUND: The periplasmic protein TolB from Escherichia coli is part of the Tol-PAL (peptidoglycan-associated lipoprotein) multiprotein complex used by group A colicins to penetrate and kill cells. TolB homologues are found in many gram-negative bacteria and the Tol-PAL system is thought to play a role in bacterial envelope integrity. TolB is required for lethal infection by Salmonella typhimurium in mice. RESULTS: The crystal structure of the selenomethionine-substituted TolB protein from E. coli was solved using multiwavelength anomalous dispersion methods and refined to 1. 95 A. TolB has a two-domain structure. The N-terminal domain consists of two alpha helices, a five-stranded beta-sheet floor and a long loop at the back of this floor. The C-terminal domain is a six-bladed beta propeller. The small, possibly mobile, contact area (430 A(2)) between the two domains involves residues from the two helices and the first and sixth blades of the beta propeller. All available genomic sequences were used to identify new TolB homologues in gram-negative bacteria. The TolB structure was then interpreted using the observed conservation pattern. CONCLUSIONS: The TolB beta-propeller C-terminal domain exhibits sequence similarities to numerous members of the prolyl oligopeptidase family and, to a lesser extent, to class B metallo-beta-lactamases. The alpha/beta N-terminal domain shares a structural similarity with the C-terminal domain of transfer RNA ligases. We suggest that the TolB protein might be part of a multiprotein complex involved in the recycling of peptidoglycan or in its covalent linking with lipoproteins.

Crystallization and preliminary crystallographic study of HIP/PAP, a human C-lectin overexpressed in primary liver cancers.

Human HIP/PAP is an adhesion protein expressed in normal pancreatic and Paneth cells and overexpressed in hepatocellular carcinoma. HIP/PAP was crystallized using the Hampton Research Crystal Screen and SAmBA software to define the optimal crystallization protocol. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 30.73, b = 49.35, c = 92.15 A and one molecule in the asymmetric unit. Flash-frozen crystals diffract to 1. 78 A resolution using synchrotron radiation. A molecular-replacement solution was obtained using the human Reg/lithostathine structure and the AMoRe software.

"Hidden" dUTPase sequence in human immunodeficiency virus type 1 gp120.

A coding region homologous to the sequence for essential eukaryotic enzyme dUTPase has been identified in different genomic regions of several viral lineages. Unlike the nonprimate lentiviruses (caprine arthritis- encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, and visna virus), where dUTPase is integrated into the pol coding region, this enzyme has never been demonstrated to be present in the primate lentivirus genomes (human immunodeficiency virus type 1 [HIV-1], HIV-2, or the related simian immunodeficiency virus). A novel approach allowed us to identify a weak but significant sequence similarity between HIV-1 gp120 and the human dUTPase. This finding was then extended to all of the primate lentivirus lineages. Together with the recently reported fragmentary structural similarity between the V3 loop region and the Escherichia coli dUTPase (P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson, Nature 393:648-659, 1998), our results strongly suggest that an ancestral dUTPase gene has evolved into the present primate lentivirus CD4 and cytokine receptor interacting region of gp120.

Crystallization and preliminary crystallographic study of a component of the Escherichia coli tol system: TolB.

TolB from Escherichia coli is part of the Tol system used by the group A colicins to penetrate and kill cells. A TolB derivative tagged with six histidines was overexpressed, purified by chelation on a nickel affinity column and crystallized using the SAmBA software to define the optimal crystallization protocol. The crystals belong to the monoclinic system, space group P21 with unit-cell parameters a = 64.48, b = 41.06, c = 78.41 A, beta = 110.78 degrees. Frozen crystals diffract to 1.9 A resolution. Screening for heavy-atom derivatives both on the native TolB and various cysteine-substituted mutants is in progress. In addition, a selenomethionine-substituted protein is being produced in order to use the MAD method for structure determination.

SAmBA: an interactive software for optimizing the design of biological macromolecules crystallization experiments.

SAmBA is a new software for the design of minimal experimental protocols using the notion of orthogonal arrays of strength 2. The main application of SAmBA is the search of protein crystallization conditions. Given a user input defining the relevant effectors/variables (e.g., pH, temperature, salts) and states (e.g., pH: 5, 6, 7 and 8), this software proposes an optimal set of experiments in which all tested variables and the pairwise interactions between them are symmetrically sampled. No a priori restrictions on the number and range of experimental variables is imposed. SAmBA consists of two complementary programs, SAm and BA, using a simulated annealing approach and a backtracking algorithm, respectively. The software is freely available as C code or as an interactive JAVA applet at http:/(/)igs-server.cnrs-mrs.fr.

Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans.

The multimeric cytochromes described to date in sulfate- and sulfur-reducing bacteria are associated with diverse respiratory modes involving the use of elemental sulfur or oxidized sulfur compounds as terminal acceptors. They exhibit no structural similarity with the other cytochrome c classes and are characterized by a bis-histidinyl axial iron coordination and low redox potentials. We have purified two new cytochromes c with markedly different molecular masses (10 000 and 50 000) from the bacterium Desulfuromonas acetoxidans, which uses anaerobic sulfur respiration as its sole energy source. The characterization by electrochemistry and optical and EPR spectroscopies revealed the cytochrome c (Mr = 10 000) to be the first monohemic cytochrome c exhibiting a bis-histidinyl axial coordination and a low redox potential (-220 mV). The cytochrome c (Mr = 50 000) contains four hemes of low potential (-200, -210, -370, and -380 mV) with the same axial coordination. The N-terminal amino acid sequences were compared with that of the trihemic cytochrome c7, previously described in D. acetoxidans and which is related to tetrahemic cytochrome c3 from sulfate reducing bacteria. Some homology was found between cytochrome c (Mr = 10 000) and cytochrome c7. Both D. acetoxidans cytochromes c are located in the periplasmic space and their biochemical and spectroscopic properties indicate that they belong to the class III cytochromes.

Identification of specificity-determining residues in antibodies.

The successful identification of the residues that contact ligand has important implications, especially in view of the increasing use of antibodies in various medical and industrial applications. Analysis of the crystallographically derived, three-dimensional structures of five antibody-antigen complexes and of the available amino acid sequence data on antibody variable regions reveals that the residues that contact antigen are in the main also the most variable. It is proposed that a good first guess of the identity of the specificity-determining residues can be made from an examination of the variability values at sequence positions. New boundaries for the complementarity-determining regions are proposed.