We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.Citrus tristeza virus (CTV) is distributed worldwide and is the causal agent of one of the most economically important diseases of citrus. CTV, a member of the genus Closterovirus within the family Closteroviridae, is phloem limited and is transmitted by aphids in a semipersistent manner. CTV virions are filamentous flexuous particles about 2,000 nm long, with two coat proteins (CP and CPm) covering 95 and 5% of the particle length, respectively (8). The CTV genome is a single-stranded, positive-sense RNA of 19,226 to 19,296 nucleotides (nt) (18,27,48,51) organized in 12 open reading frames encoding at least 19 proteins. These include two papain-like proteases, replication-associated proteins (RNA polymerase, helicase, and methyltransferase), a homologue of the HSP70 protein, two coat proteins (CP and CPm), RNA-binding protein p23 (23), a p20 protein that accumulates in the amorphous inclusion bodies (14), and other proteins of so far unknown function (p61, p13, and p18) (Fig. 1). CTV-infected plants contain, in addition to the genomic RNA, 3Ј-coterminal subgenomic RNAs (15) and defective RNAs (D RNAs), the latter resulting from extensive internal deletions of the genomic RNA (2,26,28,50).CTV isolates differing in the type and intensity of symptoms induced in different citrus species and cultivars and in their aphid transmissibility have been reported worldwide (38). In the last two decades, efforts have been taken to develop molecular techniques for rapid differentiation of CTV isolates and identification of molecular markers related to CTV-induced symptoms. Variation in serological reactivity, peptide maps of the coat protein, double-stranded RNA (dsRNA) patterns, hybridization with cDNA probes, restriction fragment length polymorphism, and single-strand conformation polymorphism (SSCP) have been described in attempts to differentiate CTV isolates (29).Nucleotide sequence analysis is the most accurate procedure for CTV differentiation and estimation of molecular or genetic variation. To date, the complete g...

The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown. The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2. We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions. Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis. No mutations affected the RNA binding capacity of the CI protein. These mutations were also introduced in the PPV genome making use of a full-length cDNA clone. Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type. These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell.

The plum pox potyvirus (PPV) cylindrical inclusion (CI) protein fused to the maltose binding protein (MBP) has been synthesized in Escherichia coli and purified by affinity chromatography in amylose resin. In the absence of any other viral factors, the fusion product had NTPase, RNA binding and RNA helicase activities. These in vitro activities were not affected by removal of the last 103 amino acids of the CI protein. However, other deletions in the C-terminal part of the protein, although leaving intact all the region conserved in RNA helicases, drastically impaired the ability to unwind dsRNA and to hydrolyze NTPs. A mutant protein lacking the last 225 residues retained the competence to interact with RNA. Further deletions mapped boundaries of the RNA binding domain within residues 350 and 402 of the PPV CI protein. This region includes the arginine-rich motif VI, the most carboxy terminal conserved domain of RNA helicases of the superfamily SF2. These results indicate that NTP hydrolysis is not an essential component for RNA binding of the PPV CI protein.

The role and different origin of brain myeloid cells in the brain is central to understanding how the central nervous system (CNS) responds to injury. C-type lectin receptor family 9, member A (DNGR-1/CLEC9A) is a marker of specific DC subsets that share functional similarities, such as CD8α(+) DCs in lymphoid tissues and CD103(+) CD11b(low) DCs in peripheral tissues. Here, we analyzed the presence of DNGR-1 in DCs present in the mouse brain (bDCs). Dngr-1/Clec9a mRNA is expressed mainly in the meningeal membranes and choroid plexus (m/Ch), and its expression is enhanced by fms-like tyrosine kinase 3 ligand (Flt3L), a cytokine involved in DC homeostasis. Using Clec9a(egfp/egfp) mice, we show that Flt3L induces accumulation of DNGR-1-EGFP(+) cells in the brain m/Ch. Most of these cells also express major histocompatibility complex class II (MHCII) molecules. We also observed an increase in specific markers of cDC CD8α+ cells such as Batf-3 and Irf-8, but not of costimulatory molecules such as Cd80 and Cd86, indicating an immature phenotype for these bDCs in the noninjured brain. The presence of DNGR-1 in the brain provides a potential marker for the study of this specific brain cell subset. Knowledge and targeting of brain antigen presenting cells (APCs) has implications for the fight against brain diseases such as neuroinflammation-based neurodegenerative diseases, microbe-induced encephalitis, and brain tumors such as gliomas.

Calcium/Calcineurin/Nuclear Factor of Activated T cells (Ca/CN/NFAT) signalling pathway is the main calcium (Ca 2+ ) dependent signalling pathway involved in the homeostasis of brain tissue. Here, we study the presence of NFATc members in human glioma by using U251 cells and a collection of primary human glioblastoma (hGB) cell lines. We show that NFATc3 member is the predominant member. Furthermore, by using constitutive active NFATc3 mutant and shRNA lentiviral vectors to achieve specific silencing of this NFATc member, we describe cytokines and molecules regulated by this pathway which are required for the normal biology of cancer cells. Implanting U251 in an orthotopic intracranial assay, we show that specific NFATc3 silencing has a role in tumour growth. In addition NFATc3 knock-down affects both the proliferation and migration capacities of glioma cells in vitro . Our data open the possibility of NFATc3 as a target for the treatment of glioma.

Amyloid precursor protein (APP) is implicated in neural development as well as in the pathology of Alzheimer's disease (AD); however, its biological function still remains unclear. It has been reported that APP stimulates the proliferation and neuronal differentiation of neural stem cells (NSCs), while other studies suggest an important effect enhancing gliogenesis in NSCs. As expected, APP protein/mRNA is detected in hNS1 cells, a model cell line of human NSCs, both under proliferation and throughout the differentiation period. To investigate the potential function that APP plays in cell fate specification and differentiation of hNS1 cells, we transiently increased human APP levels in these cells and analyzed its cell intrinsic effects. Our data indicate that increased levels of APP induce early cell cycle exit and instructively direct hNS1 cell fate towards a glial phenotype, while decreasing neuronal differentiation. Since elevated APP levels also enhanced APP intracellular domain (AICD)-immunoreactivity, these effects could be, in part, mediated by the APP/AICD system. The AICD domain can play a potential role in signal transduction by its molecular interaction with different target genes such as GSK3B, whose expression was also increased in APP-overexpressing cells that, in turn, may contribute to promoting gliogenesis and inhibiting neurogenesis in NSCs. These data suggest an important action of APP in modulating hNSCs differentiation (probably in an AICD-GSK-3β-dependent manner) and may thus be important for the future development of stem cell therapy strategies for the diseased mammalian brain.

Background Aging and age-related diseases are strong risk factors for the development of neurodegenerative diseases. Neuroinflammation (NIF), as the brain’s immune response, plays an important role in aged associated degeneration of central nervous system (CNS). There is a need for well characterized animal models that will allow the scientific community to understand and modulate this process. Methods We have analyzed aging-phenotypical and inflammatory changes of brain myeloid cells (bMyC) in a senescent accelerated prone aged (SAMP8) mouse model, and compared with their senescence resistant control mice (SAMR1). We have performed morphometric methods to evaluate the architecture of cellular prolongations and determined the appearance of Iba1+ clustered cells with aging. To analyze specific constant brain areas, we have performed stereology measurements of Iba1+ cells in the hippocampal formation. We have isolated bMyC from brain parenchyma (BP) and choroid plexus plus meningeal membranes (m/Ch), and analyzed their response to systemic lipopolysaccharide (LPS)-driven inflammation. Results Aged 10 months old SAMP8 mice present many of the hallmarks of aging-dependent neuroinflammation when compared with their SAMR1 control, i.e., increase of protein aggregates, presence of Iba1+ clusters, but not an increase in the number of Iba1+ cells. We have further observed an increase of main inflammatory mediator IL-1β, and an augment of border MHCII+Iba1+ cells. Isolated CD45+ bMyC from brain parenchyma (BP) and choroid plexus plus meningeal membranes (m/Ch) have been analyzed, showing that there is not a significant increase of CD45+ cells from the periphery. Our data support that aged-driven pro-inflammatory cytokine interleukin 1 beta (IL-1β) transcription is enhanced in CD45+BP cells. Furthermore, LPS-driven systemic inflammation produces inflammatory cytokines mainly in border bMyC, sensed to a lesser extent by the BP bMyC, showing that IL-1β expression is further augmented in aged SAMP8 compared to control SAMR1. Conclusion Our data validate the SAMP8 model to study age-associated neuroinflammatory events, but careful controls for age and strain are required. These animals show morphological changes in their bMyC cell repertoires associated to age, corresponding to an increase in the production of pro-inflammatory cytokines such as IL-1β, which predispose the brain to an enhanced inflammatory response after LPS-systemic challenge.

In recent years, our knowledge of the molecular biology of plum pox potyvirus (PPV) and of potyviruses in general has greatly increased. The genome of PPV consists of a unique single‐strand RNA molecule with messenger polarity. A terminal protein (VPg) and a poly‐(A) tail are present at the 5’and 3’ends, respectively, of the genomic RNA. Four complete and other partial nucleotide sequences of PPV isolates have been reported. According to the levels of homology three different PPV strains can be defined. PPV RNA is translated into a unique polyprotein starting at the second AUG codon of the large open‐reading frame that covers most of the genome. The polyprotein is proteolytically processed by three virus encoded proteases (P1Pro, HCPro and NIaPro). While P1Pro and HCPro cut at their own carboxyl termini, NIaPro recognizes seven cleavage sites characterized by conserved heptapeptides. These sites differ in their susceptibility to cis and trans processing and in their reaction profiles. Immunoelectron microscopy has shown NIa and NIb proteins to form nuclear inclusions, but also dense aggregates in the cytoplasm, and CI protein to make typical pinwheel cytoplasmic inclusions. On the basis of sequence comparisons, NIb protein has been proposed to be the viral RNA replicase. CI protein has been purified from infected leaves and from Escherichia coli harbouring a plasmid that encodes it. It has nucleic acid‐stimulated NTPase and RNA helicase activities. Virus infection has been achieved by inoculation with a PPV full‐length cDNA clone and with transcripts synthesized using another one as template. These clones can be used to apply genetic engineering techniques to the in vitro manipulation of the PPV genome.