The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis.

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.

Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei.

Activation of the p34cdc2 protein kinase (PK) at different stages of the eukaryotic cell cycle is controlled by interaction with regulatory proteins known as cyclins (CYCs). Using a probe obtained by PCR amplification, we have isolated from the protozoan, Trypanosoma brucei, a cDNA clone encoding a CYC homologue. The amino acid sequence deduced for this gene (CYC1) shares structural homology with A- and B-type CYCs of other organisms, including a motif, the destruction box, which has been related to the rapid turnover of these CYC proteins in mitosis. When expressed in fission yeast, CYC1 is able to rescue the defect of a temperature-sensitive cdc13 mutant, demonstrating that it is functional as a cell-cycle regulator. In trypanosome cells, CYC1 associates with a 34-kDa protein that cross-reacts with a monoclonal antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the complex displays histone H1 PK activity. Furthermore, when trypanosome cells are synchronized by hydroxyurea treatment, CYC1 accumulates as cells progress towards mitosis. These observations, taken together, suggest that CYC1 is a component of the active PK complex required for the control of trypanosome mitosis.

The expression of the major shed Trypanosoma cruzi antigen results from the developmentally-regulated transcription of a small gene family.

To better understand the mechanisms involved in the developmental expression of Trypanosoma cruzi antigens we examined the gene structure and transcription properties of the major shed trypomastigote antigen (SAPA). We report in this paper that SAPA is encoded by a small family of at lest 6 genes which differ mainly in the length of a repeat region made up of tandemly arranged 36-bp repeat units. SAPA genes are located distant from chromosomal telomeres as inferred from their insensitivity to Bal31 nuclease treatment. Furthermore, Northern blot and S1 protection analyses strongly support the fact that most (or all) SAPA genes are transcribed in the infective form of the parasite.

Variable number of repeat units in genes encoding Trypanosoma cruzi antigens.

The genes encoding a protein antigenic during the acute phase (SAPA) and another one antigenic during the chronic phase (antigen 30) of human Chagas' disease were analyzed in 15 Trypanosoma cruzi isolates and clones collected in distant geographical regions. These two genes had tandem repeats which were present in all parasites tested. However, large differences in the length of restriction fragments were observed among isolates. This was readily explained by variations in the number of repeat units present in homologous genes. This result was confirmed after analysis of 3 members of the SAPA gene family. In the case of antigen 30, we propose that differences in the number of repeat units result in size differences in the corresponding RNAs and proteins, which explains the large size heterogeneity in otherwise homologous T. cruzi antigens.

Antigenic determinants of Trypanosoma cruzi defined by cloning of parasite DNA.

A genomic DNA library from Trypanosoma cruzi, the agent of Chagas' disease, was constructed in the gt11 lambda vector and was screened with serum from a Chagasic patient. Out of 53 positive clones, 23 plaques were purified to homogeneity and 10 different groups were defined by cross-hybridization experiments and by reaction of antibodies selected with products from each recombinant clone. Native T. cruzi proteins of molecular mass ranging from 85 to larger than 205 kDa that share antigenic determinants with products of the recombinant clones were observed in Western blots of parasite extracts. Some of the native proteins were detected in the trypomastigote stage of the parasite, while others were present in epimastigotes as well. The latter result was confirmed for some recombinant clones by hybridization of the cloned DNA with Northern blots of parasite RNA. Clones from each group reacted differently with nine sera from rabbits infected with several T. cruzi strains as well as with eight sera from human patients. Clone 7 was detected by all rabbit sera but not by three human sera. Conversely, clones 1, 2 and 30 were detected by all human sera but failed to be detected by most rabbit sera. We conclude that several proteins from T. cruzi are antigenically active during infection and that some of them differ in their ability to generate antibodies in rabbit or human infections.

Respiratory control in mitochondria from Trypanosoma cruzi.

Phosphorylating mitochondrial membranes were obtained from Trypanosoma cruzi culture (epimastigote) forms. Using ADP as phosphate acceptor and succinate, sn-glycerol-3-phosphate, L-malate or ascorbate plus tetramethyl-p-phenylenediamine (TMPD) as oxidizable substrates, energy coupling sites II and III were detected, with respiratory control values in the range of 2.8-2.0. Carbonyl cyanide m-chlorophenylhydrazone and sonication uncoupled the respiratory control mechanism. Antimycin and cyanide partially inhibited succinate, sn-glycerol-3-phosphate and L-malate oxidation, while cyanide totally inhibited ascorbate + TMPD oxidation by the mitochondrial preparation. Succinate oxidation was inhibited by malonate and oxalacetate, but this latter was only effective with sonicated mitochondria. At variance with other substrates, NADH oxidation was not controlled by ADP concentration or inhibited by antimycin or cyanide. Rotenone failed to inhibit electron transfer in T. cruzi mitochondria.

Polymorphisms within minicircle sequence classes in the kinetoplast DNA of Trypanosoma cruzi clones.

Four minicircle classes were analyzed using cloned minicircles as probes and single-cell cloned Trypanosoma cruzi parasites. The hybridization conditions used allowed identification of minicircle classes within kinetoplast DNA that were non-homologous to each other. Two of these minicircle classes, detected with probes pTckAWP-2 and -3, were present together in several of the CA 1 and Miranda clones, in spite of the fact that either pTckAWP-2 or both minicircle classes were undetectable in other isolates and clones of the parasite. The other two minicircle classes (pTckM-84 and -88) were located in some Miranda cloned parasites which were characterized by the simple restriction endonuclease pattern of their minicircles. Both pTckM-84 and -88 minicircle classes represented 52-71% of the kinetoplast DNA in the latter group of trypanosomes. Restriction endonuclease mapping allowed the identification of polymorphic minicircles in two of the four minicircle classes analyzed (pTckAWP-2 and pTckM-88). The polymorphisms were observed in part of the molecules of one minicircle class within a single trypanosome clone, as well as when different clones or even some of those obtained from the same isolate were compared. In addition, a different proportion of pTckM-88 type of minicircle sequence class was observed in the kinetoplast DNA from two of the Miranda clones analyzed. These observations demonstrated that similar molecules may evolve independently in sequence in each parasite. The polymorphic minicircles detected may arise from sequence variations before expansion of a future homogeneous minicircle sequence class.

Multiple Trypanosoma cruzi antigens containing tandemly repeated amino acid sequence motifs.

Chromosomal DNA from Trypanosoma cruzi, the agent of the American trypanosomiasis (Chagas' disease), was used for construction of a DNA library, employing the expression vector lambda gt11. Nine clones encoding different parasite antigens were isolated from this library by screening with an antiserum from a Chagasic patient. Nucleotide sequence analysis showed that seven out of the nine isolated clones code for antigens which contain tandemly repeated amino acid sequence motifs. Each of the seven antigens contains a unique repeat, ranging in length between 5 and 68 amino acids. The length of the repeats is highly conserved within each clone. Fusion proteins, expressed from two of the clones, reacted with a large proportion of sera collected from Chagasic patients in Argentina, Brazil and Chile. These clones appear thus to encode antigens which are shared between different strains of T. cruzi. Immunofluorescence experiments with live parasites showed that three of the antigens were detectable on the surface of trypanosomes.

Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease.

A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.

Mutational analysis of the feline immunodeficiency virus matrix protein.

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.

Small variations in the length of the cytoplasmic domain of the simian immunodeficiency virus transmembrane protein drastically affect envelope incorporation and virus entry.

Simian immunodeficiency viruses (SIVs) have an envelope (Env) glycoprotein with an unusually long cytoplasmic domain of 164 amino acids. In this article, we have characterized a series of SIV Env truncation mutants in which the cytoplasmic domain was progressively shortened from its carboxyl terminus by 20 amino acids. Expression by means of the vaccinia virus system showed that all of the SIV Env mutants were expressed and processed into the surface and transmembrane (TM) subunits. When the ability of the Env mutants to associate with SIV Gag particles was examined, we found that deletion of 20 to 80 residues from the carboxyl terminus of the SIV TM cytoplasmic tail abrogated the incorporation of the Env glycoprotein into particles. By contrast, further truncation of the SIV TM protein by 100 to 140 amino acids restored the ability of the Env protein to associate with Gag particles. Interestingly, mutants bearing a 44- or 24-amino acid cytoplasmic domain were incorporated at levels significantly higher than those of the wild-type Env. Single-cycle infectivity assays showed that Env mutants bearing cytoplasmic tails of 144 to 64 amino acids were highly inefficient at mediating virus entry. By contrast, truncation of the cytoplasmic domain to 44 or 24 amino acids drastically enhanced virus infectivity with respect to that conferred by the full-length Env protein. Our results demonstrate that small variations in the length of the SIV Env cytoplasmic domain dramatically influence Env-mediated viral functions.

[Existence of 2 sites of oxidative phosphorylation in Trypanosoma cruzi]. / Existencia de dos sitios de fosforilación oxidativa en Trypanosoma cruzi.

Phosphorylating mitochondrial preparations were obtained from T. cruzi culture (epimastigote) forms by grinding the cells with glass - beads and differential centrifugation. Using ADP as phosphate acceptor and succinate, L-malate or ascorbate + tetramethyl-p-phenylenediamine (TMPD) as oxidizable substrates, the respiratory control (R.C.) values were (mean +/- S.D.; n = 4, in parenthesis, the substrate): 2.8 +/- 0.10 (succinate): 2.3 +/- 0.13 (L-malate) and 2.0 +/- 0.12 (ascorbate + TMPD). The ADP:O values were 1.68 +/- 0.08, 1.42 +/- 0.08 and 0.66 +/- 0.66 +/- 0.12, respectively. The uncoupler CCCP stimulated substrate oxidation somewhat more than ADP. Succinate oxidation was by malonate and also by oxaloacetate but the latter was effective only after sonicating the mitochondrial preparation. The mitochondrial membranes oxidized also NADH but this oxidation was not subjected to control by the phosphate acceptor. Our results support the existence of two energy-conserving sites in T. cruzi respiratory chain (sites 2 and 3) and confirm previous observations by Stoppani et al. (Mol. Biochem. Parasitol. 2:3-21, 1980) with intact epimastigotes.

[Cytochromes in different stages, strains, and populations of Trypanosoma cruzi]. / Citocromos en diferentes estadios, cepas y poblaciones de Trypanosoma cruzi.

Differential reduced minus oxidized (Red-Ox) or reduced. CO minus reduced (Red. CO-Red) spectra of Trypanosoma cruzi metacyclic trypomastigotes (Tulahuen strain), revealed the presence of cytochromes aa3, b, c5 5 8, o and possibly d (Fig. 1). Mitochondrial membranes from the epimastigote form of the parasite showed similar cytochrome spectra (Fig. 2A). Extraction of the mitochondrial membranes with guanidine and cholate, and spectroscopy of the cytochrome o -cyanide derivative proved that this cytochrome is an integral constituent of the mitochondrial membranes (Figs. 2B and 4, and Table 1). Contamination of the investigated samples with hemoglobin, peroxidase, catalase, cytochrome P-420, cytochrome P-450 or culture medium hemoproteins was ruled out by filtering the cells and mitochondrial membranes on Sephadex G-50, or by differential spectroscopy of pigments, or by differential centrifugations of samples. Investigation of pyridine-hemochromes revealed hemes A, B, C and D (Fig. 3), thus confirming the presence of the postulated cytochromes. Comparative spectroscopy of a series of T. cruzi stocks (including Tulahuen and Y strains), many of them obtained from acute or chronic forms of Chagas disease, revealed significant variability in the cytochrome content (Table 2). Taking cytochromes o and b as standard for comparison, the epimastigotes samples could be grouped as follows (in parenthesis number of passages through the culture medium): 1) stocks with a relatively high content of cytochromes b and o, prevailing the former (stocks Y (116), RA (114), AF, FN, TN and MG (14 y 16); 2) stocks with a relatively low content of both cytochromes: Y (119), AWP and UP; 3) stocks with a low content of cytochrome b, without cytochrome o: CA-I and CA-I (V); 4) stocks without cytochromes: Y(117 and 118) and RA(113). In some strains (e.g. Y and RA), significant variation of cytochrome content in different stocks of the same isolate was observed (Table 2), but the Tulahuen strain proved to be less variable. Comparison of cytochrome distribution and other properties of parasites, namely, lethality for mice and morphology, did not allow to establish positive correlations.

Mutational analysis of the conserved cysteine residues in the simian immunodeficiency virus matrix protein.

The matrix protein (MA) of human and simian immunodeficiency viruses (HIV and SIV) is encoded by the amino-terminal region of the Gag precursor and has been suggested to be involved in different processes during the early and late stages of the virus life cycle. The MA protein of SIV contains three cysteine residues at positions 57, 83, and 87, which are also highly conserved among HIV-2 isolates. In order to study the functional significance of these residues in virus morphogenesis, a series of mutations affecting the cysteines of SIV MA were introduced into a gag-protease construct and expressed in the vaccinia vector system. The MA mutants were assayed for their ability to synthesize and process the Gag polyprotein precursor as well as to release particles into the culture medium. In addition, the incorporation of the envelope glycoprotein (Env) into the Gag-made particles was investigated. Substitution of alanine for cysteine 87 had little effect on particle release and Env glycoprotein association. By contrast, the individual replacement of cysteines 57 or 83 by alanine, as well as the simultaneous mutation of cysteines 83 and 87, significantly reduced the ability of Gag polypeptides to produce extracellular particles. Assembly into particles appeared to be also affected, albeit to a lesser extent, when both cysteines 57 and 83 were replaced by alanine. Furthermore, substitution of cysteine 83 in the SIV MA domain was found to be detrimental to Gag polyprotein processing. Analysis of the Env glycoprotein association with recombinant particles revealed that this process was moderately affected in the case of the double mutants lacking cysteines 57 and 83, or cysteines 57 and 87, and the cysteine-minus triple mutant. Our results suggest that the conserved cysteines 57 and 83 in the MA domain are important for efficient SIV Gag particle production.

Assembly of double-layered virus-like particles in mammalian cells by coexpression of human rotavirus VP2 and VP6.

Development in mammalian cells of a recombinant expression system that mimics the rotavirus capsid assembly process would be advantageous for studying the structural requirements for particle formation. To this end, we investigated the ability of a recombinant vaccinia virus system to produce double-layered virus-like particles. The genes coding for VP2 and VP6 proteins of the human rotavirus strain Wa were cloned and used to generate recombinant vaccinia viruses. Metabolic labelling of CV-1 cells infected with these recombinant viruses followed by immunoprecipitation with a polyclonal antiserum directed to Wa virus showed that VP2 and VP6 were efficiently expressed. The recombinant proteins were similar in size and immunoreactivity to authentic rotavirus proteins. Biochemical and electron microscopy analyses demonstrated that simultaneous expression of VP2 and VP6 in mammalian cells resulted in the formation of intracellular spherical particles resembling double-layered rotavirus particles.

Deletion mapping of functional domains in the rotavirus capsid protein VP6.

VP6, the major capsid protein of rotavirus, oligomerizes into trimers that constitute the intermediate shell of the virions. In order to map functional domains in this protein, we introduced seven internal in-frame deletions within the coding region of gene 6 of human rotavirus strain Wa. Regions of homology among the VP6 proteins of group A and group C rotaviruses were targeted for deletion mutagenesis. The mutant VP6 proteins were expressed in mammalian cells using the recombinant vaccinia virus system and were examined for their ability to oligomerize into trimers as well as to assemble into double-layered virus-like particles upon coexpression with the rotavirus core protein VP2. Deletions that abolished trimerization defined a domain (residues 246 to 314) that maps within a larger region previously found to be critical for oligomerization (amino acids 105 to 328). When the capacity of each mutant to assemble into double-layered virus-like particles was analysed, three different assembly phenotypes were observed. Phenotype I was represented by two deletion mutants lacking residues 246 to 250 and 308 to 314 that produced particles with efficiencies similar to that of wild-type VP6. Phenotype II, characterized by a moderate decrease in the efficiency of particle assembly with respect to that of wild-type VP6, included two mutants with deletions at the C terminus of the protein. Phenotype III was exhibited by three mutants whose abilities to assemble into double-layered virus-like particles were drastically impaired. Two of these mutants define a previously unidentified assembly domain (amino acids 122 to 147) at the N terminus of rotavirus VP6.

Substitution of leucine 8 in the simian immunodeficiency virus matrix protein impairs particle formation without affecting N-myristylation of the Gag precursor.

In the late stages of replication of the simian immunodeficiency viruses (SIV), the matrix protein (MA) plays a central role in the transport of Pr55gag to the plasma membrane, assembly of virus particles, and incorporation of the envelope glycoprotein into particles. Targeting of Pr55gag to the plasma membrane is mediated by two motifs within the MA protein: the N-terminal myristate and a cluster of positively charged amino acids. In this report, we characterized the assembly phenotype of an SIV Gag mutant (L8Q) carrying the single amino acid substitution of glutamine for leucine at position 8 in the MA domain. The hydropathic profile of the mutated MA protein indicated that the L8Q amino acid change disrupts the hydrophobic character of the region comprising the first 10 residues of the protein. Expression of mutant L8Q Gag protein in CV-1 cells, by means of the vaccinia virus vector system, resulted in efficient synthesis and N-myristylation of Pr55gag. However, this mutation severely impaired particle production, as inferred from both biochemical and electron microscopy analyses. Cellular fractionation assays revealed that in cells expressing mutant L8Q, the proportion of cytosol-associated Pr55gag was significantly increased compared to that observed upon expression of wild-type Gag. Furthermore, mutant L8Q Gag partitioned onto cytosol and membrane fractions in a manner similar to nonmyristylated Gag polyprotein. Taken together, these results indicate that the L8Q mutation reduces the membrane-binding capacity of the Gag precursor. It is therefore likely that in the SIV MA, in addition to the N-myristate group, the hydrophobicity of the neighboring region is important for efficient association of Pr55gag with the plasma membrane.

Expression of biologically active envelope glycoprotein from the acutely pathogenic simian immunodeficiency virus SIVsmmPBj.

The full-length envelope (env) gene from the most acutely pathogenic primate lentivirus described so far, the simian immunodeficiency virus SIVsmmPBj14 was expressed by a recombinant vaccinia virus vector (vv-env4) and was completely characterized as a previous step for its use as an immunogen in vaccination trials. Radioimmunoprecipitation and Western blot experiments indicated that SIVsmmPBj gp160 precursor was processed into gp120 and gp41 subunits, and that gp120 was released into the medium. Flow cytometry analysis showed that recombinant SIVsmmPBj was transported to and expressed on the surface of vvenv4-infected cells. Biochemical analysis of virus-like particles produced by coinfection of cells with recombinant vaccinia viruses expressing SIVsmmPBj Env (vv-env4) and Gag (vv-wtgag) proteins revealed that the Env glycoprotein was incorporated into core-like particles. Furthermore, cells expressing SIVsmmPBj env gene products were found to undergo fusion with the same CD4+ cell lines in which the whole provirus has been shown to form syncytia.

Identification of domains in the simian immunodeficiency virus matrix protein essential for assembly and envelope glycoprotein incorporation.

The matrix domain (MA) of the simian immunodeficiency virus (SIV) is encoded by the amino-terminal region of the Gag polyprotein precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. To define domains of the SIV MA protein that are involved in virus morphogenesis, deletion and substitution mutations were introduced in this protein in the context of a gag-protease construct and expressed in the vaccinia virus vector system. The MA mutants were characterized with respect to synthesis and processing of the Gag precursor, assembly and release of virus-like particles, and incorporation of the envelope (Env) glycoprotein into particles. We have identified two regions of the SIV MA which are critical for particle formation. Both domains are located in a central hydrophobic alpha-helix of the SIV MA, according to data on the structure of this protein. In addition, we have characterized a domain whose mutation impairs the incorporation of SIV Env glycoproteins with long transmembrane cytoplasmic tails into particles. Interestingly, these mutant particles retained the ability to associate with SIV Env proteins with short cytoplasmic tails.