Developing therapeutically more efficient Neurturin variants for treatment of Parkinson's disease.

In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue.

Protein secretome of moss plants (Physcomitrella patens) with emphasis on changes induced by a fungal elicitor.

Studies on extracellular proteins (ECPs) contribute to understanding of the multifunctional nature of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte.

Genomics and Proteomics Provide New Insight into the Commensal and Pathogenic Lifestyles of Bovine- and Human-Associated Staphylococcus epidermidis Strains.

The present study reports comparative genomics and proteomics of Staphylococcus epidermidis (SE) strains isolated from bovine intramammary infection (PM221) and human hosts (ATCC12228 and RP62A). Genome-level profiling and protein expression analyses revealed that the bovine strain and the mildly infectious ATCC12228 strain are highly similar. Their genomes share high sequence identity and synteny, and both were predicted to encode the commensal-associated fdr marker gene. In contrast, PM221 was judged to differ from the sepsis-associated virulent human RP62A strain on the basis of distinct protein expression patterns and overall lack of genome synteny. The 2D DIGE and phenotypic analyses suggest that PM221 and ATCC12228 coordinate the TCA cycle activity and the formation of small colony variants in a way that could result in increased viability. Pilot experimental infection studies indicated that although ATCC12228 was able to infect a bovine host, the PM221 strain caused more severe clinical signs. Further investigation revealed strain- and condition-specific differences among surface bound proteins with likely roles in adhesion, biofilm formation, and immunomodulatory functions. Thus, our findings revealed a close link between the bovine and commensal-type human strains and suggest that humans could act as a reservoir of bovine mastitis-causing SE strains.

Human single-chain urokinase is activated by the omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.

Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in omptin proteolysis.

Regulation of AT1R expression through HuR by insulin.

Angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. Type 2 diabetes is hyperinsulinemic state and a major risk factor for atherosclerosis and hypertension. It is known that hyperinsulinemia upregulates AT1R expression post-transcriptionally by increasing the half-life of AT1R mRNA, but little is known about the mechanism of this effect. In the present study, we first identified AT1R 3'-UTR as a mediator of insulin effect. Using 3'-UTR as a bait, we identified through analysis of insulin-stimulated cell lysates by affinity purification and mass spectrometry HuR as an insulin-regulated AT1R mRNA binding protein. By ribonucleoprotein immunoprecipitation, we found HuR binding to AT1R to be increased by insulin. Overexpression of HuR leads to increased AT1R expression in a 3'-UTR-dependent manner. Both insulin and HuR overexpression stabilize AT1R 3'-UTR and their responsive element within 3'-UTR are located within the same region. Cell fractionation demonstrated that insulin induced HuR translocation from nucleus to cytoplasm increased HuR binding to cytoplasmic AT1R 3'-UTR. Consistent with HuR translocation playing a mechanistic role in HuR effect, a reduction in the cytoplasmic levels of HuR either by silencing of HuR expression or by inhibition of HuR translocation into cytoplasm attenuated insulin response. These results show that HuR translocation to cytoplasm is enhanced by insulin leading to AT1R upregulation through HuR-mediated stabilization of AT1R mRNA.

Characterization of the genome, proteome, and structure of yersiniophage ϕR1-37.

The bacteriophage vB_YecM-ϕR1-37 (ϕR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage ϕR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of ϕR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the ϕR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage ϕKZ. The tail of ϕR1-37 has a contractile sheath. We conclude that phage ϕR1-37 is a representative of a novel phage type that carries the dU-containing genome in a ϕKZ-like head.

Structural determinants of vascular endothelial growth factor-D receptor binding and specificity.

Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2-specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function.

Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo.

In Parkinson's disease, brain dopamine neurons degenerate most prominently in the substantia nigra. Neurotrophic factors promote survival, differentiation and maintenance of neurons in developing and adult vertebrate nervous system. The most potent neurotrophic factor for dopamine neurons described so far is the glial-cell-line-derived neurotrophic factor (GDNF). Here we have identified a conserved dopamine neurotrophic factor (CDNF) as a trophic factor for dopamine neurons. CDNF, together with its previously described vertebrate and invertebrate homologue the mesencephalic-astrocyte-derived neurotrophic factor, is a secreted protein with eight conserved cysteine residues, predicting a unique protein fold and defining a new, evolutionarily conserved protein family. CDNF (Armetl1) is expressed in several tissues of mouse and human, including the mouse embryonic and postnatal brain. In vivo, CDNF prevented the 6-hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons in a rat experimental model of Parkinson's disease. A single injection of CDNF before 6-OHDA delivery into the striatum significantly reduced amphetamine-induced ipsilateral turning behaviour and almost completely rescued dopaminergic tyrosine-hydroxylase-positive cells in the substantia nigra. When administered four weeks after 6-OHDA, intrastriatal injection of CDNF was able to restore the dopaminergic function and prevent the degeneration of dopaminergic neurons in substantia nigra. Thus, CDNF was at least as efficient as GDNF in both experimental settings. Our results suggest that CDNF might be beneficial for the treatment of Parkinson's disease.

Proteomics and transcriptomics characterization of bile stress response in probiotic Lactobacillus rhamnosus GG.

Lactobacillus rhamnosus GG (GG) is a widely used and intensively studied probiotic bacterium. Although the health benefits of strain GG are well documented, the systematic exploration of mechanisms by which this strain exerts probiotic effects in the host has only recently been initiated. The ability to survive the harsh conditions of the gastrointestinal tract, including gastric juice containing bile salts, is one of the vital characteristics that enables a probiotic bacterium to transiently colonize the host. Here we used gene expression profiling at the transcriptome and proteome levels to investigate the cellular response of strain GG toward bile under defined bioreactor conditions. The analyses revealed that in response to growth of strain GG in the presence of 0.2% ox gall the transcript levels of 316 genes changed significantly (p < 0.01, t test), and 42 proteins, including both intracellular and surface-exposed proteins (i.e. surfome), were differentially abundant (p < 0.01, t test in total proteome analysis; p < 0.05, t test in surfome analysis). Protein abundance changes correlated with transcriptome level changes for 14 of these proteins. The identified proteins suggest diverse and specific changes in general stress responses as well as in cell envelope-related functions, including in pathways affecting fatty acid composition, cell surface charge, and thickness of the exopolysaccharide layer. These changes are likely to strengthen the cell envelope against bile-induced stress and signal the GG cells of gut entrance. Notably, the surfome analyses demonstrated significant reduction in the abundance of a protein catalyzing the synthesis of exopolysaccharides, whereas a protein dedicated for active removal of bile compounds from the cells was up-regulated. These findings suggest a role for these proteins in facilitating the well founded interaction of strain GG with the host mucus in the presence of sublethal doses of bile. The significance of these findings in terms of the functionality of a probiotic bacterium is discussed.

Structural determinants of growth factor binding and specificity by VEGF receptor 2.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

Identification and characterization of gushing-active hydrophobins from Fusarium graminearum and related species.

Fungal infection of barley and malt, particularly by the Fusarium species, is a direct cause of spontaneous overfoaming of beer, referred to as gushing. We have shown previously that small fungal proteins, hydrophobins, act as gushing-inducing factors in beer. The aim of our present study was to isolate and characterize hydrophobins from a gushing-active fungus, Fusarium graminearum (teleomorph Gibberella zeae) and related species. We generated profile hidden Markov models (profile HMMs) for the hydrophobin classes Ia, Ib and II from the multiple sequence alignments of their known members available in public domain databases. We searched the published Fusarium graminearum genome with the Markov models. The best matching sequences and the corresponding genes were isolated from F. graminearum and the related species F. culmorum and F. poae by PCR and characterized. One each of the putative F. graminearum and F. poae hydrophobin genes were expressed in the heterologous host Trichoderma reesei. The proteins corresponding to the genes were purified and identified as hydrophobins and named GzHYD5 and FpHYD5, respectively. Concentrations of 0.003 ppm of these hydrophobins were observed to induce vigorous beer gushing.

Actin and RIG-I/MAVS signaling components translocate to mitochondria upon influenza A virus infection of human primary macrophages.

Influenza A virus is one of the most important causes of respiratory infection. During viral infection, multiple cell signaling cascades are activated, resulting in the production of antiviral cytokines and initiation of programmed cell death of virus-infected cells. In the present study, we have used subcellular proteomics to reveal the host response to influenza A infection at the protein level in human macrophages. Macrophages were infected with influenza A virus, after which the cytosolic and mitochondrial cell fractions were prepared and analyzed by using two-dimensional electrophoresis for protein separation and mass spectrometry for protein identification. In cytosolic proteomes, the level of several heat shock proteins and fragments of cytoskeletal proteins was clearly up-regulated during influenza A virus infection. In mitochondrial proteomes, simultaneously with the expression of viral proteins, the level of intact actin and tubulin was highly up-regulated. This was followed by translocation of the components of antiviral RNA recognition machinery, including RIG-I (retinoic acid-inducible protein I), TRADD (TNFR1-associated death domain protein), TRIM25 (tripartite motif protein 25), and IKKepsilon (inducible IkappaB kinase), onto the mitochondria. Cytochalasin D, a potent inhibitor of actin polymerization, clearly inhibited influenza A virus-induced expression of IFN-beta, IL-29, and TNF-alpha, suggesting that intact actin cytoskeleton structure is crucial for proper activation of antiviral response. At late phases of infection mitochondrial fragmentation of actin was seen, indicating that actin fragments, fractins, are involved in disruption of mitochondrial membranes during apoptosis of virus-infected cells. In conclusion, our results suggest that actin network interacts with mitochondria to regulate both antiviral and cell death signals during influenza A virus infection.

Posttranscriptional regulation of angiotensin II type 1 receptor expression by glyceraldehyde 3-phosphate dehydrogenase.

Regulation of angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. We started from an observation that the 3'-untranslated region (3'-UTR) of AT1R mRNA suppressed AT1R translation. Using affinity purification for the separation of 3'-UTR-binding proteins and mass spectrometry for their identification, we describe glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an AT1R 3'-UTR-binding protein. RNA electrophoretic mobility shift analysis with purified GAPDH further demonstrated a direct interaction with the 3'-UTR while GAPDH immunoprecipitation confirmed this interaction with endogenous AT1R mRNA. GAPDH-binding site was mapped to 1-100 of 3'-UTR. GAPDH-bound target mRNAs were identified by expression array hybridization. Analysis of secondary structures shared among GAPDH targets led to the identification of a RNA motif rich in adenines and uracils. Silencing of GAPDH increased the expression of both endogenous and transfected AT1R. Similarly, a decrease in GAPDH expression by H(2)O(2) led to an increased level of AT1R expression. Consistent with GAPDH having a central role in H(2)O(2)-mediated AT1R regulation, both the deletion of GAPDH-binding site and GAPDH overexpression attenuated the effect of H(2)O(2) on AT1R mRNA. Taken together, GAPDH is a translational suppressor of AT1R and mediates the effect of H(2)O(2) on AT1R mRNA.

The omptins of Yersinia pestis and Salmonella enterica cleave the reactive center loop of plasminogen activator inhibitor 1.

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.

Human L-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl Lewis x in core 2 O-glycans.

Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO(3)) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLe(x)). TyrSO(3) at position Y118 was more critical for binding than at Y97. C2-SLe(x) at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLe(x). P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO(3) residues in association with a C2-SLe(x) moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.

An ssDNA virus infecting archaea: a new lineage of viruses with a membrane envelope.

Archaeal organisms are generally known as diverse extremophiles, but they play a crucial role also in moderate environments. So far, only about 50 archaeal viruses have been described in some detail. Despite this, unusual viral morphotypes within this group have been reported. Interestingly, all isolated archaeal viruses have a double-stranded DNA (dsDNA) genome. To further characterize the diversity of archaeal viruses, we screened highly saline water samples for archaea and their viruses. Here, we describe a new haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1) that was isolated from a solar saltern and infects an indigenous host belonging to the genus Halorubrum. Infection does not cause cell lysis, but slightly retards growth of the host and results in high replication of the virus. The sequenced genome (7048 nucleotides) of HRPV-1 is single-stranded DNA (ssDNA), which makes HRPV-1 the first characterized archaeal virus that does not have a dsDNA genome. In spite of this, similarities to another archaeal virus were observed. Two major structural proteins were recognized in protein analyses, and by lipid analyses it was shown that the virion contains a membrane. Electron microscopy studies indicate that the enveloped virion is pleomorphic (approximately 44 x 55 nm). HRPV-1 virion may represent commonly used virion architecture, and it seems that structure-based virus lineages may be extended to non-icosahedral viruses.

Deficiency of the INCL protein Ppt1 results in changes in ectopic F1-ATP synthase and altered cholesterol metabolism.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disease caused by deficiency of palmitoyl protein thioesterase 1 (PPT1). INCL results in dramatic loss of thalamocortical neurons, but the disease mechanism has remained elusive. In the present work we describe the first interaction partner of PPT1, the F(1)-complex of the mitochondrial ATP synthase, by co-purification and in vitro-binding assays. In addition to mitochondria, subunits of F(1)-complex have been reported to localize in the plasma membrane, and to be capable of acting as receptors for various ligands such as apolipoprotein A-1. We verified here the plasma membrane localization of F(1)-subunits on mouse primary neurons and fibroblasts by cell surface biotinylation and TIRF-microscopy. To gain further insight into the Ppt1-mediated properties of the F(1)-complex, we utilized the Ppt1-deficient Ppt1(Delta ex4) mice. While no changes in the mitochondrial function could be detected in the brain of the Ppt1(Delta ex4) mice, the levels of F(1)-subunits alpha and beta on the plasma membrane were specifically increased in the Ppt1(Delta ex4) neurons. Significant changes were also detected in the apolipoprotein A-I uptake by the Ppt1(Delta ex4) neurons and the serum lipid composition in the Ppt1(Delta ex4) mice. These data indicate neuron-specific changes for F(1)-complex in the Ppt1-deficient cells and give clues for a possible link between lipid metabolism and neurodegeneration in INCL.

Recombination and selection pressure in the ipomovirus sweet potato mild mottle virus (Potyviridae) in wild species and cultivated sweetpotato in the centre of evolution in East Africa.

Sweet potato mild mottle virus (SPMMV) is the type member of the genus Ipomovirus (family Potyviridae). SPMMV occurs in cultivated sweetpotatoes (Ipomoea batatas Lam.; Convolvulaceae) in East Africa, but its natural wild hosts are unknown. In this study, SPMMV was detected in 283 (9.8 %) of the 2864 wild plants (family Convolvulaceae) sampled from different agro-ecological zones of Uganda. The infected plants belonged to 21 species that were previously not known to be natural hosts of SPMMV. The size of the SPMMV coat protein (CP) was determined by Western blot analysis, N-terminal protein sequencing and peptide mass fingerprinting. Data implicated a proteolytic cleavage site, VYVEPH/A, at the NIb/CP junction, resulting in a CP of approximately 35 kDa. Nearly complete sequences of 13 SPMMV isolates were characterized. Phylogenetic analysis of non-recombinant CP-encoding sequences placed five isolates from wild species sampled in the central zone of Uganda into a separate cluster. Recombination events were detected in the 5'- and 3'-proximal parts of the genome, providing novel evidence of recombination in the genus Ipomovirus. Thirteen amino acids in the N terminus of the P1 protein were under positive selection, whereas purifying selection was implicated for the HC-Pro-, P3-, 6K1- and CP-encoding regions. These data, supported by previous studies on ipomoviruses, provide indications of an evolutionary process in which the P1 proteinase responds to the needs of adaptation.

The closest relatives of icosahedral viruses of thermophilic bacteria are among viruses and plasmids of the halophilic archaea.

We have sequenced the genome and identified the structural proteins and lipids of the novel membrane-containing, icosahedral virus P23-77 of Thermus thermophilus. P23-77 has an approximately 17-kb circular double-stranded DNA genome, which was annotated to contain 37 putative genes. Virions were subjected to dissociation analysis, and five protein species were shown to associate with the internal viral membrane, while three were constituents of the protein capsid. Analysis of the bacteriophage genome revealed it to be evolutionarily related to another Thermus phage (IN93), archaeal Halobacterium plasmid (pHH205), a genetic element integrated into Haloarcula genome (designated here as IHP for integrated Haloarcula provirus), and the Haloarcula virus SH1. These genetic elements share two major capsid proteins and a putative packaging ATPase. The ATPase is similar with the ATPases found in the PRD1-type viruses, thus providing an evolutionary link to these viruses and furthering our knowledge on the origin of viruses.

p100 increases AT1R expression through interaction with AT1R 3'-UTR.

p100 protein (SND1, Tudor-SN) is a multifunctional protein that functions as a co-activator for several transcription factors, has a role in mRNA processing and participates in RNAi-induced silencing complex (RISC) with yet unknown function. In this study we identified a novel function for p100 as a regulator of angiotensin II type 1 receptor (AT1R) expression. The binding of p100 to AT1R 3'-untranslated region (3'-UTR) via staphylococcal nuclease-like (SN-like) domains increased receptor expression by decreasing the rate of mRNA decay and enhancing its translation. Overexpression of p100 increased AT1R expression, whereas decrease in p100 binding to 3'-UTR either by p100 silencing or by the deletion of p100 binding site downregulated receptor expression. The effect of p100 through AT1R 3'-UTR was independent of Argonaute2 (Ago2), a known p100 partner, and was thus RISC-independent. Nucleotides 118 to 120 of the AT1R 3'-UTR were found to be critical for the binding of p100 to 3'-UTR. In summary, p100 is a multifunctional regulator of gene expression that regulates transcription, mRNA maturation, and as described in this article, also mRNA stability and translation.