Nathalie Chamond

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3

Translation initiation on HIV genomic RNA relies on both cap and Internal Ribosome Entry Site (IRES) dependant mechanisms that are regulated throughout the cell cycle. During a unique phenomenon, the virus recruits initiation complexes through RNA structures located within Gag coding sequence, downstream of the initiation codon. We analyzed initiation complexes paused on the HIV-2 gag IRES and revealed that they contain all the canonical initiation factors except eIF4E and eIF1. We report that eIF3 and the small ribosomal subunit bind HIV RNA within gag open reading frame. We thus propose a novel two step model whereby the initial event is the formation of a ternary eIF3/40S/IRES complex. In a second step, dependent on most of the canonical initiation factors, the complex is rearranged to transfer the ribosome on the initiation codons. The absolute requirement of this large structure for HIV translation defines a new function for a coding region. Moreover, the level of information compaction within this viral genome reveals an additional level of evolutionary constraint on the coding sequence. The conservation of this IRES and its properties in rapidly evolving viruses suggest an important role in the virus life cycle and highlight an attractive new therapeutic target.

A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA

Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5′ untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIVMac coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role.

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity.

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect‐transmitted protozoan responsible for Chagas’ disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B‐cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non‐secreted isoform of proline racemase by an alternative trans‐splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock‐down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite‐induced B‐cell polyclonal activation.

Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. I. Parasitological, hematological and pathological parameters.

African trypanosomiasis is a severe parasitic disease that affects both humans and livestock. Several different species may cause animal trypanosomosis and although Trypanosoma vivax (sub-genus Duttonella) is currently responsible for the vast majority of debilitating cases causing great economic hardship in West Africa and South America, little is known about its biology and interaction with its hosts. Relatively speaking, T. vivax has been more than neglected despite an urgent need to develop efficient control strategies. Some pioneering rodent models were developed to circumvent the difficulties of working with livestock, but disappointedly were for the most part discontinued decades ago. To gain more insight into the biology of T. vivax, its interactions with the host and consequently its pathogenesis, we have developed a number of reproducible murine models using a parasite isolate that is infectious for rodents. Firstly, we analyzed the parasitical characteristics of the infection using inbred and outbred mouse strains to compare the impact of host genetic background on the infection and on survival rates. Hematological studies showed that the infection gave rise to severe anemia, and histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with extramedullary hematopoiesis in the liver, and cerebral edema. The models developed are consistent with field observations and pave the way for subsequent in-depth studies into the pathogenesis of T. vivax - trypanosomosis.

Selection and Characterization of RNA Interference-Deficient Trypanosomes Impaired in Target mRNA Degradation

ABSTRACT Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with α-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei. Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).

In vitro studies reveal that different modes of initiation on HIV-1 mRNA have different levels of requirement for eukaryotic initiation factor 4F.

Expression of the two isoforms p55 and p40 of HIV‐1 Gag proteins relies on distinct translation initiation mechanisms, a cap‐dependent initiation and two internal ribosome entry sites (IRESs). The regulation of these processes is complex and remains poorly understood. This study was focused on the influence of the 5′‐UTR and on the requirement for the eukaryotic initiation factor (eIF)4F complex components. By using an in vitro system, we showed substantial involvement of the 5′‐UTR in the control of p55 expression. This highly structured 5′‐UTR requires the eIF4F complex, especially RNA helicase eIF4A, which mediates initiation at the authentic AUG codon. In addition, the 5′‐UTR regulates expression in an RNA concentration‐dependent manner, with a high concentration of RNA triggering specific reduction of full‐length Gag p55 production. HIV‐1 genomic RNA also has the ability to use a strong IRES element located in the gag coding region. We show that this mechanism is particularly efficient, and that activity of this IRES is only poorly dependent on RNA helicase eIF4A when the viral 5′‐UTR is removed. HIV‐1 genomic mRNA exhibits in vitro translational features that allow the expression of Gag p55 protein by different mechanisms that involve different requirements for eIF4E, eIF4G, and eIF4A. This suggests that HIV‐1 could adapt to its mode of translation according to the availability of the initiation factors in the infected cell.

RNA Interference in Trypanosoma brucei ROLE OF THE N-TERMINAL RGG DOMAIN AND THE POLYRIBOSOME ASSOCIATION OF ARGONAUTE

Argonaute proteins (AGOs) are central to RNA interference (RNAi) and related silencing pathways. At the core of the RNAi pathway in the ancient parasitic eukaryote Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in the destruction of potentially harmful retroposon transcripts. One notable feature of TbAGO1 is that a fraction sediments with polyribosomes, and this association is facilitated by an arginine/glycine-rich domain (RGG domain) at the N terminus of the protein. Here we report that reducing the size of the RGG domain and, in particular, mutating all arginine residues severely reduced the association of TbAGO1 with polyribosomes and RNAi-induced cleavage of mRNA. However, these mutations did not change the cellular localization of Argonaute and did not affect the accumulation of single-stranded siRNAs, an essential step in the activation of the RNA-induced silencing complex. We further show that mRNA on polyribosomes can be targeted for degradation, although this alliance is not a pre-requisite. Finally, sequestering tubulin mRNAs from translation with antisense morpholino oligonucleotides reduced the RNAi response indicating that mRNAs not engaged in translation may be less accessible to the RNAi machinery. We conclude that the association of the RNAi machinery and target mRNA on polyribosomes promotes an efficient RNAi response. This mechanism may represent an ancient adaptation to ensure that retroposon transcripts are efficiently destroyed, if they become associated with the translational apparatus.

Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. II. Immunobiological dysfunctions.

Trypanosoma vivax is the main species involved in trypanosomosis, but very little is known about the immunobiology of the infective process caused by this parasite. Recently we undertook to further characterize the main parasitological, haematological and pathological characteristics of mouse models of T. vivax infection and noted severe anemia and thrombocytopenia coincident with rising parasitemia. To gain more insight into the organisms immunobiology, we studied lymphocyte populations in central (bone marrow) and peripherical (spleen and blood) tissues following mouse infection with T. vivax and showed that the immune system apparatus is affected both quantitatively and qualitatively. More precisely, after an initial increase that primarily involves CD4+ T cells and macrophages, the number of splenic B cells decreases in a step-wise manner. Our results show that while infection triggers the activation and proliferation of Hematopoietic Stem Cells, Granulocyte-Monocyte, Common Myeloid and Megacaryocyte Erythrocyte progenitors decrease in number in the course of the infection. An in-depth analysis of B-cell progenitors also indicated that maturation of pro-B into pre-B precursors seems to be compromised. This interferes with the mature B cell dynamics and renewal in the periphery. Altogether, our results show that T. vivax induces profound immunological alterations in myeloid and lymphoid progenitors which may prevent adequate control of T. vivax trypanosomosis.

40S recruitment in the absence of eIF4G/4A by EMCV IRES refines the model for translation initiation on the archetype of Type II IRESs

Initiation of translation on Type II IRESs, such as those of EMCV and FMDV viruses, has been well documented in the recent years. For EMCV, the current model argues for a mechanism in which the key interaction necessary for the pre-initiation complex recruitment is eIF4G binding to the central J-K domains of EMCV-IRES. Here we demonstrate that, in contrast with the current model, the molecular mechanism of EMCV-IRES involves direct recruitment of the 40S subunit. Importantly, we identified a specific structural element that prevents the correct positioning of the initiation codon in the close vicinity of the ribosomal P site. This work clarifies how this interaction could not be anticipated by earlier studies and allows us to propose a new model for initiation complex assembly on EMCV-IRES. The role attributed to eIF4G/4A can thus be refined as stabilizing/promoting the conformational changes that are necessary for IRES function, thus resembling the role conventionally assigned to ITAFs. This raises the interesting possibility that IRESs are primarily ribosome binders, some of which having partly lost the ability to fold into the active structure without the help of proteins.