Infectious vaccine-derived rubella viruses emerge, persist, and evolve in cutaneous granulomas of children with primary immunodeficiencies.

Rubella viruses (RV) have been found in an association with granulomas in children with primary immune deficiencies (PID). Here, we report the recovery and characterization of infectious immunodeficiency-related vaccine-derived rubella viruses (iVDRV) from diagnostic skin biopsies of four patients. Sequence evolution within PID hosts was studied by comparison of the complete genomic sequences of the iVDRVs with the genome of the vaccine virus RA27/3. The degree of divergence of each iVDRV correlated with the duration of persistence indicating continuous intrahost evolution. The evolution rates for synonymous and nonsynonymous substitutions were estimated to be 5.7 x 10-3 subs/site/year and 8.9 x 10-4 subs/site/year, respectively. Mutational spectra and signatures indicated a major role for APOBEC cytidine deaminases and a secondary role for ADAR adenosine deaminases in generating diversity of iVDRVs. The distributions of mutations across the genes and 3D hotspots for amino acid substitutions in the E1 glycoprotein identified regions that may be under positive selective pressure. Quasispecies diversity was higher in granulomas than in recovered infectious iVDRVs. Growth properties of iVDRVs were assessed in WI-38 fibroblast cultures. None of the iVDRV isolates showed complete reversion to wild type phenotype but the replicative and persistence characteristics of iVDRVs were different from those of the RA27/3 vaccine strain, making predictions of iVDRV transmissibility and teratogenicity difficult. However, detection of iVDRV RNA in nasopharyngeal specimen and poor neutralization of some iVDRV strains by sera from vaccinated persons suggests possible public health risks associated with iVDRV carriers. Detection of IgM antibody to RV in sera of two out of three patients may be a marker of virus persistence, potentially useful for identifying patients with iVDRV before development of lesions. Studies of the evolutionary dynamics of iVDRV during persistence will contribute to development of infection control strategies and antiviral therapies.

Three-dimensional models of human 2'-5' oligoadenylate synthetases: a new computational method for reconstructing an enzyme assembly.

BACKGROUND: 2'-5' Oligoadenylate synthetases are interferon-induced enzymes important for antiviral cell defense. Tetramer formation of human OAS1 is essential for the catalytic activity of the enzyme. However, no structure of any oligomeric OAS1 protein has yet been solved and the structural organization of the catalytically active tetramers is not known. MATERIAL/METHODS: Using a novel conjunction of comparative modeling, molecular surface analysis, and sequence analysis, high-resolution models of the known isoforms of human OAS1 were prepared. RESULTS: The resulting models suggest different oligomeric states for the p40, p46, and p48 isoforms as well as a significant difference in enzymatic activity between the p46 and p40/p48 isoforms. CONCLUSIONS: The differences in enzymatic activity could result in different susceptibility to viral infection in cells expressing individual isoforms. The models are consistent with the published biochemical data on human OAS1. The results also suggest that the structure of the active OAS2 dimers would be equivalent to that of p40 OAS1 tetramers.

Computed energetics of nucleotides in spatial ribozyme structures: an accurate identification of functional regions from structure.

Ribozymes are functionally diverse RNA molecules with intrinsic catalytic activity. Multiple structural and biochemical studies are required to establish which nucleotide bases are involved in the catalysis. The relative energetic properties of the nucleotide bases have been analyzed in a set of the known ribozyme structures. It was found that many of the known catalytic nucleotides can be identified using only the structure without any additional biochemical data. The results of the calculations compare well with the available biochemical data on RNA stability. Extensive in silico mutagenesis suggests that most of the nucleotides in ribozymes stabilize the RNA. The calculations show that relative contribution of the catalytic bases to RNA stability observably differs from contributions of the noncatalytic bases. Distinction between the concepts of "relative stability" and "mutational stability" is suggested. As results of prediction for several models of ribozymes appear to be in agreement with the published data on the potential active site regions, the method can potentially be used for prediction of functional nucleotides from nucleic sequence.

Analysis of protein structures reveals regions of rare backbone conformation at functional sites.

Regions of rare conformation were located in 300 protein crystal structures representing seven major protein folds. A distance matrix algorithm was used to search rapidly for 9-residue fragments of rare backbone conformation using a comparison to a relational database of encoded fragments derived from the database of nonredundant structures. Rare fragments were found in 61% of the analyzed protein structures. Detailed analysis was performed for 78 proteins of different folds. The rare fragments were located near functional sites in 72% of the protein structures. The rare fragments often formed parts of ligand-binding sites (59%), protein-protein interfaces (8%), and domain-domain contacts (5%). Of the remaining structures, 5% had a high average B-factor or high local B-factors. Statistical analysis suggests that the association between ligands and rare regions does not occur by chance alone. The present study is likely to underestimate the number of functional sites, because not all analyzed protein structures contained a ligand. The results suggest that rapid searches for regions with rare local backbone conformations can assist in prediction of functional sites in novel proteins.

Protein folding: search for basic physical models.

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context of in vivo protein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in the in vitro folding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

Close pairs of carboxylates: a possibility of multicenter hydrogen bonds in proteins.

Covalent attachment of hydrogen to the donor atom may be not an essential characteristic of stable hydrogen bonds. A positively charged particle (such as a proton), located between the two negatively charged residues, may lead to a stable interaction of the two negative residues. This paper analyzes close Asp-Glu pairs of residues in a large set of protein chains; 840 such pairs of residues were identified, of which 28% were stabilized by a metal ion, 12% by a positive residue nearby and 60% are likely to be stabilized by a proton. The absence of apparent structural constraints, secondary structure preferences, somewhat lower B-factors and a distinct correlation between pH and the minimal O-O distance in carboxylate pairs suggest that most of the abnormally close pairs could indeed be stabilized by a shared proton. Implications for protein stability and modeling are discussed.

Structural criteria of biologically active RGD-sites for analysis of protein cellular function - a bioinformatics study.

BACKGROUND: Cell adhesion involves interactions of integrins and extracellular proteins, often facilitated by the RGD motif. Only presence of the RGD in a sequence of a protein may be not sufficient for the biological activity (binding to an integrin) and additional biochemical and/or structural studies are essential. MATERIAL/METHODS: Structural criteria that would allow identification biologically active RGD-sites on the base of a spatial structure may assist analysis of function of a protein in the cell. For the first time, computational analysis of RGD-sites in a large non-redundant set of protein structures was done. RESULTS: Out of 3819 protein chains sequences of about 100 contained RGDs. Analysis of the structures of the RGD-'native' proteins has allowed establishing main determinants of the biologically active conformations of the RGD sites: surface accessibility of the whole RGD-sequence and the secondary structure. The criteria, applied to the remaining proteins of the set, identify 23 proteins ( approximately 25%) with potentially active RGD-sites. The results strongly suggest that RGD has a high propensity for being involved in protein-protein interactions and this may explain occurrence of RGDs in intracellular proteins. Results of the analysis suggest (in some cases, confirm) novel integrin-related activities for 7 membrane/extracellular proteins, as well as confirm RGD-facilitated cell attachment for 5 viral proteins. CONCLUSIONS: Only presence of RGD in a sequence is not sufficient to propose biological activity of this site. The results also suggest that the method can be used on large scale: for example, for identifying potential integrin-interacting proteins in an animal genome.

Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sites.

The HIV-1 proteinase (PR) has proved to be a good target for antiretroviral therapy of AIDS, and various PR inhibitors are now in clinical use. However, there is a rapid selection of viral variants bearing mutations in the proteinase that are resistant to clinical inhibitors. Drug resistance also involves mutations of the nucleocapsid/p1 and p1/p6 cleavage sites of Gag, both in vitro and in vivo. Cleavages at these sites have been shown to be rate limiting steps for polyprotein processing and viral maturation. Furthermore, these sites show significant sequence polymorphism, which also may have an impact on virion infectivity. We have studied the hydrolysis of oligopeptides representing these cleavage sites with representative mutations found as natural variations or that arise as resistant mutations. Wild-type and five drug resistant PRs with mutations within or outside the substrate binding site were tested. While the natural variations showed either increased or decreased susceptibility of peptides toward the proteinases, the resistant mutations always had a beneficial effect on catalytic efficiency. Comparison of the specificity changes obtained for the various substrates suggested that the maximization of the van der Waals contacts between substrate and PR is the major determinant of specificity: the same effect is crucial for inhibitor potency. The natural nucleocapsid/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis. Hence, mutation of these rate limiting cleavage sites can partly compensate for the reduced catalytic activity of drug resistant mutant HIV-1 proteinases.

Geometric criteria of hydrogen bonds in proteins and identification of "bifurcated" hydrogen bonds.

Empirical criteria for identification of hydrogen bonds were analyzed to produce a set of geometrically consistent criteria. For a data set of 30 structures, application of a set of purely geometrical criteria, along with exclusion of abnormal backbone conformations, also excluded a common interaction of Ser/Thr side chains with Asp/Glu side chains ([ST]/[DE] pairs). These interactions were termed "bifurcated hydrogen bonds", which implies delocalization of a positively charged hydrogen of hydroxyl between the two acceptor atoms of the carboxylic group. These "bifurcated" interactions are among the most common packing patterns for [ST]/[DE] pairs of side chains. Therefore, the identification of hydrogen bonds cannot be based on geometrical criteria only and requires introduction of some physico-chemical criteria.

Functional maps of the junctions between interglobular contacts and active sites in glycolytic enzymes -- a comparative analysis of the biochemical and structural data.

BACKGROUND: Oligomers and separate subunits of the glycolytic enzymes often have different catalytic properties. However, spectral data show an apparent lack of significant conformational changes during oligomerization. Since the conformation of an enzyme determines its catalytic properties, the structural mechanism(s) influencing the activity is of considerable interest. MATERIAL/METHODS: Analysis of the spatial structures of the junctions between interglobular contacts and binding sites may give a clue to the mechanism(s) of the activation. In this work, the problem was studied using available structural and biochemical data for the oligomeric enzymes of glycolysis. RESULTS: Computational analysis of the structures of the junctions has identified three structurally distinct types of junctions: 1. interglobular binding site (2 of 8 enzymes); 2. domain-domain stabilization (5 of 8); and 3. 'sequence overlap' or a local conformational change (all enzymes). Thus the catalytic activity may be influenced through the shifts of the modules of protein structure (types 1, 2) and/or due to a slight change in the local structure (type 3). The more common junctions of types 2 and 3 are well conserved among eukaryotic enzymes, which suggests their biological importance. CONCLUSIONS: The results suggest that a profound and a complex change in conformation in subunits of an oligomeric enzyme may not be necessary for a significant change in the catalytic properties. The analysis maps the residues important for the junctions and thus for the link between the catalytic activity and the oligomeric state of the enzymes.

Crystal structures of Tcl1 family oncoproteins and their conserved surface features.

Members of the TCL1 family of oncogenes are abnormally expressed in mature T-cell leukemias and B-cell lymphomas. The proteins are involved in the coactivation of protein kinase B (Akt/PKB), a key intracellular kinase. The sequences and crystal structures of three Tcl1 proteins were analyzed in order to understand their interactions with Akt/PKB and the implications for lymphocyte malignancies. Tcl1 proteins are approximately 15 kD and share 25-80% amino acid sequence identity. The tertiary structures of mouse Tcl1, human Tcl1, and Mtcp1 are very similar. Analysis of the structures revealed conserved semi-planar surfaces that have characteristics of surfaces involved in protein-protein interactions. The Tcl1 proteins show differences in surface charge distribution and oligomeric state suggesting that they do not interact in the same way with Akt/PKB and other cellular protein(s).