Véronique Mayau

Nonpathogenic SIV infection of African green monkeys induces a strong but rapidly controlled type I IFN response

African green monkeys (AGMs) infected with the AGM type of SIV (SIVagm) do not develop chronic immune activation and AIDS, despite viral loads similar to those detected in humans infected with HIV-1 and rhesus macaques (RMs) infected with the RM type of SIV (SIVmac). Because chronic immune activation drives progressive CD4+ T cell depletion and immune cell dysfunctions, factors that characterize disease progression, we sought to understand the molecular basis of this AGM phenotype. To this end, we longitudinally assessed the gene expression profiles of blood- and lymph node-derived CD4+ cells from AGMs and RMs in response to SIVagm and SIVmac infection, respectively, using a genomic microarray platform. The molecular signature of acute infection was characterized, in both species, by strong upregulation of type I IFN-stimulated genes (ISGs). ISG expression returned to basal levels after postinfection day 28 in AGMs but was sustained in RMs, especially in the lymph node-derived cells. We also found that SIVagm induced IFN-alpha production by AGM cells in vitro and that low IFN-alpha levels were sufficient to induce strong ISG responses. In conclusion, SIV infection triggered a rapid and strong IFN-alpha response in vivo in both AGMs and RMs, with this response being efficiently controlled only in AGMs, possibly as a result of active regulatory mechanisms.

Identification of a family of Rab G-proteins in Plasmodium falciparum and a detailed characterisation of pfrab6

As a first step towards developing a set of compartment-specific probes for studying protein trafficking in the malaria-infected erythrocyte, we describe here a family of Plasmodium falciparum Rab proteins. We characterise in detail P. falciparum Rab6 (PfRab6) a marker which in other cells is specific for the Golgi/trans Golgi network. Although PfRab6 mRNA is expressed throughout the intraerythrocytic cycle, maximal expression occurs at the trophozoite stage. Immunofluorescence microscopy shows that the distribution of PfRab6 changes during the final stages of parasite maturation, coalescing into multiple foci, each of which is associated with the nucleus of a forming daughter parasite.

Gag p27-Specific B- and T-Cell Responses in Simian Immunodeficiency Virus SIVagm-Infected African Green Monkeys

ABSTRACT Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4+ and CD8+ T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.

Long oligonucleotide microarrays for African green monkey gene expression profile analysis

Nonhuman primates, including African green monkey (AGM), are important models for bio‐medical research. The information on monkey genomes is still limited and no versatile gene expression screening tool is available. We tested human whole genome microarrays for cross‐species reactivity with AGM transcripts using both long oligonucleotide arrays (60‐mer probes) and short oligonucleotide arrays (25‐mer). Using the long oligonucleotide arrays, we detected 4‐fold more AGM transcripts than with the short oligonucleotide technology. The number of detected transcripts was comparable to that detected using human RNA, with 87% of the detected genes being shared between both species. The specificity of the signals obtained with the long oligonucleotide arrays was determined by analyzing the transcriptome of concanava‐lin A‐activated CD4+ T cells vs. nonactivated T cells of two monkey species AGM and macaque. For both species, the genes showing the most significant changes in expression, such as IL‐2R, were those known to be regulated in human CD4+ T cell activation. Finally, tissue specificity of the signals was established by comparing the transcription profiles of AGM brain and tonsil cells. In conclusion, the ABI human microarray platform provides a highly valuable tool for the assessment of AGM gene expression profiles.—Jacquelin B., Mayau, V., Brysbaert, G., Regnault, B., Diop, O. M., Arenzana‐Seisdedos, F., Rogge, L., Coppée J‐Y., Barré‐Sinoussi F., Benecke, A., Müller‐Trutwin M. C. Long oligonucleotide microarrays for African green monkey gene expression profile analysis. FASEB J. 21, 3262–3271 (2007)

Characterization of the unprocessed and processed forms of rab6 expressed in baculovirus/insect cell systems

Rab6 protein (rab6p) belongs to a family of ras-like GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. We have constructed a recombinant baculovirus in order to express rab6p in insect cells. We report here the characterization of four forms of this protein which are found in cytosolic and membrane fractions of infected Sf9 cells. The two major forms are a cytosolic 24 kD protein which represents the unprocessed precursor form of rab6p and a membrane-bound isoprenylated 23 kD protein which represents the processed form. Two other minor forms were also detected: a cytosolic isoprenylated 23 kD protein which may represent a pool in equilibrium with the 23 kD membrane-bound form and a 24 kD non-isoprenylated membrane-bound form which may represent an intermediate in the processing of rab6p.

Twelve genes, including the unassigned proteasome ζ subunit gene, ordered within the human 1p13 region

Using a coamplification mapping technique, we previously established the map of a cluster of genes located on Chinese hamster Chromosome (Chr) lq. It was shown that the gene coding the adenosine monophosphate deaminase2 (AMPD2) is flanked on one side by members of the glutathione S-transferase p. family genes (GSTM), and on the other side by the genes encoding the c t2 and the csi3 subunits of the G proteins (GNAT2 and GNAI3 respectively). The GNAI3, GNAT2, AMPD2, and one member of the GSTMs genes were found clustered, in this relative order, within 100 kb in the Chinese hamster genome (Kellems 1992). The direction of transcription of these genes was determined and the sizes of two intergenic regions (GNAI3—GNAT2 and GNAT2AMPD2) were also precisely defined (Baron et al. 1996). Further away, on the side of the GNAI3 gene, were found the genes encoding a Seryl-tRNA Synthetase (SARS; Kellems 1992) and the C subunit of the proteasome. This latter gene has been identified by comparing the partial sequence of a 750-bp hamster cDNA clone to the sequences deposited in the GDB sequence library. A high level of homology was found with the human PSMA5 gene and the rat homolog (unpublished results). To determine whether the Chinese hamster gene order is conserved in the human genome, we screened the YAC library from the CEPH laboratory by PCR, using GNAI3 specific primers (Table 1), and seven positive clones were selected for further analysis. Oligonucleotide pairs were constructed to amplify sequences specific for genes potentially linked to the GNAI3 gene. The sequence of some primers was obtained from published data, and we designed the others from sequences available in the GDB sequence library (Table 1). Each pair of primers was used to amplify target sequences from total genomic DNA recovered from the seven selected YAC clones, from Hela cells as a positive control, and from normal yeast and the unrelated YAC clone 950F6 as negative controls. The experiments have been repeated at least three times, and reproducible results were obtained (Table 2). We found that the AMPD2 and the GNAT2 (primers described in GDB: GOO 376-941) genes are present on all the YACs positive for the GNAI3 gene. This result strongly suggests a tight linkage of the three genes, but it does not allow one to determine their order in the human genome. The fact that most other genes were present only on a subset of the studied YACs permitted generation of a physical map of the region (Table 2). As in the Chinese hamster genome, the SARS and the PSMA5 genes were found on one side of the three genes mentioned above, and their relative order was established. On the other side of the three central genes, we identified the five members of the GSTM multigenic family, using previously described gene-specific primer sets (Pearson et al. 1993). In good agreement with this work, we found that the genes are clustered. The GSTM4 and GSTM2 genes lie close to the GNAI3, GNAl2, and AMPD2 group of genes. GSTM1, M3, and M5 were altogether present or absent on the studied YACs and

Inflammatory control in AIDS-resistant non human primates

African non human primates are natural hosts of SIV. The infection is non-pathogenic despite plasma viral load levels similar to those in HIV-1 infected humans and SIVmac-infected macaques (MAC) progressing towards AIDS. The most striking difference between non-pathogenic SIV and pathogenic HIV-1/SIVmac infections is the lack of chronic T cell activation in natural hosts. In HIV and SIVmac infections, chronic T cell activation is known to drive CD4+T cell depletion. Intense research efforts are worldwide put on the search of the mechanisms that can control chronic T cell activation in HIV/SIV infections. Innate immune responses play a determinant role in the regulation of T cell activation profiles. Type I interferons (IFN-I) are part of the first-wave response of the innate immune system in viral infections. We compared the IFN-I responses between pathogenic (MAC) and non-pathogenic SIV infections (African Green monkey, AGM) at the level of blood and lymph nodes (LN) during the early and chronic stage of infection. During the acute SIVagm infection, we detected high amounts of IFN-α in the plasma of AGMs, although the mean levels at the peak were three times lower than in MAC. The microarray data revealed a rapid and strong up-regulation of type I Interferon-Stimulated Genes (ISG) in AGMs during acute SIVagm infection. ISGs denote the in vivo activity of IFN-I. Using a functional assay, we demonstrated that low IFN-α concentrations (50 times lower than the IFN-α levels in plasma at the peak) were sufficient to induce strong ISG responses in AGM and MAC cells. Surprisingly, our direct comparison of blood and LNs showed that ISG induction was broader in blood of AGMs than in MAC, while in LN, it was the contrary. Thus, in AGMs, less ISG were induced in LNs as compared to MAC already during the acute phase of infection. Moreover, our tight kinetic analysis showed that this ISG expression was efficiently controlled after day 28 post-infection in AGMs, while in MAC the ISGs expression remained uncontrolled. Finally, we identified genes that were differentially expressed between the two species and which might be involved in the discriminating responses. Altogether, this shows that AGMs are capable to mount a well coordinated and efficient regulative response to innate immune activation.