Self-renewal of murine embryonic stem cells is supported by the serine/threonine kinases pim-1 and pim-3

International audiencePim-1 and pim-3 encode serine/threonine kinases involved in the regulation of cell proliferation and apoptosis in response to cytokine stimulation. We analyzed the regulation of pim-1 and pim-3 by the leukemia inhibitory factor (LIF)/gp130/signal transducer and activator of transcription-3 (STAT3) pathway and the role of Pim-1 and Pim-3 kinases in mouse embryonic stem (ES) cell self-renewal. Making use of ES cells expressing a granulocyte colony-stimulating factor: gp130 chimeric receptor and a hormone-dependent signal transducer and activator of transcription-3 estrogen receptor (STAT3-ERT2), we showed that expression of pim-1 and 3 was upregulated by LIF/gp130-dependent signaling and the STAT3 transcription factor. ES cells overexpressing pim-1 and pim-3 had a greater capacity to self-renew and displayed a greater resistance to LIF starvation based on a clonal assay. In contrast, knockdown of pim-1 and pim-3 increased the rate of spontaneous differentiation in a self-renewal assay. Knockdown of pim-1 and pim-3 was also detrimental to the growth of undifferentiated ES cell colonies and increased the rate of apoptosis. These findings provide a novel role of Pim-1 and Pim-3 kinases in the control of self-renewal of ES cells

Characterization of hepatitis C virus particle subpopulations reveals multiple usage of the scavenger receptor BI for entry steps

Hepatitis C virus (HCV) particles assemble along the very low density lipoprotein pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities. Little is known about the cell entry pathway of these different HCV particle subpopulations, which likely occurs by regulated spatiotemporal processes involving several cell surface molecules. One of these molecules is the scavenger receptor BI (SR-BI), a receptor for high density lipoprotein that can bind to the HCV glycoprotein E2. By studying the entry properties of infectious virus subpopulations differing in their buoyant densities, we show that these HCV particles utilize SR-BI in a manifold manner. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apolipoprotein E, present on the surface of HCV particles, but not the E2 glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important entry factors such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscore a third function of SR-BI governed by specific residues in hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2

A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism.

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses

BIS analysis of dengue E-Pr coevolving residues.

<p>(<b>A</b>) Tridimensional representation of DENV Pr (Black, PDB 3C6R) and E (multi-color, PDB 1K4R). A linear representation of the PrM-E polyprotein is depicted below the protein structures. Starting and ending residue positions of each protein (Pr, M and E) and E domain are indicated. E domains are annotated by distinct colors: DI, domain I (red); DII, domain II (yellow); DIII, domain III (blue); Tmd, transmembrane (black). (<b>B</b>) Organization and positions of the PrM-E cluster 2 (orange), 7 (blue) and 9 (pink) on tridimensional representations of the DENV E and Pr proteins. Cluster 2 and 9 are depicted on a dimeric or trimeric E-Pr structure respectively at low pH condition (PDB 3C6R). Cluster 7 is depicted on a trimeric E-Pr structure at neutral pH condition (PDB 3XIY). Linear representation of the two proteins are also depicted on the top of each structure and cluster block location are indicated (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s003" target="_blank">S1 Table</a></b>). The close proximity between DENV E and Pr cluster 2 blocks is enlarged.</p

BIS as a methodology to decrypt virus entry mechanisms.

<p>Schematic representation of the experimental approach employed in this study, from BIS computational analysis to the design and challenge of a mechanistic model of viral fusion. Following sequence analysis, matrix of E1E2 amino-acid coevolution were generated by BIS for different HCV genotypes. Plotting of matrix coevolution networks onto E2core structure unveiled a potential scenario of E1 and E2 rearrangements during HCV fusion, which involved the BL domain of E2. At the protein domain level, the construction of a soluble form of the BL and the conduction of several experimental assays supported such hypothesis. In parallel, at the amino acid level, the experimental validation of coevolution signals between specific residues of E1 and of the BL highlighted the critical role of E1-BL networks in regulating fusogenic rearrangements (and more generally, the critical role of coevolving networks between E1 and E2 C-terminal regions). Altogether, this approach allows us to propose a HCV fusion model where BL movements and E1 refolding are critical in the induction of E1E2 interdependent, fusogenic rearrangements. By being applicable to other viral proteins and viruses, such approach provides opportunities to uncover undescribed viral-mediated mechanisms and design innovative translational strategies for their inhibition.</p

An E2 BLd-derived peptide inhibits HCV infection.

<p>(<b>A</b>) Putative BL movements that can modulate E2 rearrangements within E1E2 heterodimers. Rotation angles of the E2core structure are indicated (Reference: <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g003" target="_blank">Fig 3B</a></b>) and illustrated by a black cross harboring 4 color extremities. (<b>B</b>) Schematic representation of the E2 core structure and of the two hypothetical E2 conformational states prior (stretched) and following (packed) virus fusion when E1E2 are associated as heterodimer (color code and angle of E2 structures refer to <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g003" target="_blank">Fig 3B</a></b>). (<b>C</b>) Schematic representation of the expression construct encoding for the BLd-H77 peptide derived from the gt1a H77 E2 glycoprotein. SP, signal peptide; 6H, 6His-tag. (<b>D</b>) Detection of BLd-H77 in non-reducing condition following SDS-Page electrophoresis and coomassie blue staining. Molecular weight of the reference ladder are indicated on the left (kDa). (<b>E</b>) Detection of BLd-H77 peptide by Western Blot using a 6His-tag antibody. A 6His-tagged soluble E2 was used as a positive control. (<b>F</b>) Dose-dependent inhibition of HCVcc infection. HCVcc H77/JFH-1 or JC1 were pre-incubated with different doses of BLd-H77 (μg/ml) and used to infect Huh7.5 cells. Percentages of primary infection were calculated according to the viral titers of HCVcc particles incubated without BLd-H77 (mean ± SD; n = 3). (<b>G</b>) Kinetic of action of BLd-H77 on HCVcc infection. Huh7.5 cells were incubated 1h with BLd-H77 prior infection (35μg/ml) of HCVcc H77/JFH-1 (2), during the 4h infection (3) or following infection (secondary infection; 4). As control, cells were incubated at each step with equivalent volume of PBS (1). Percentages of primary infection were calculated according to viral titers of control conditions (mean ± SD; n = 3). Statistical significances <i>(</i>*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001) were determined for each experimental condition versus control condition (100%). (<b>H</b>) Dose-dependent inhibition of HCVpp harboring H77 envelope (HCVpp-H77) or control pseudoparticles harboring VSV-G (VSVGpp) envelope glycoprotein. Pseudoparticles were pre-incubated with different doses of BLd-H77 (μg/ml) or with PBS and used to infect Huh7.5 cells. Percentage of GFP positive cells was determined and expressed as percentages of infection, according to the viral titers of HCVpp incubated with PBS (mean ± SD; n = 3). (<b>I</b>) Kinetic of action of BLd-H77 on HCVpp infection. Huh7.5 cells were incubated 1h with BLd-H77 prior HCVpp H77 infection (50μg/ml) (2), during the 4h infection (3) or following infection (4). As control, cells were incubated at each step with equivalent volume of PBS (1). Percentages of infection according to viral titers of control conditions are reported (mean ± SD; n = 3). Statistical significances <i>(</i>**<i>p</i><0.01, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>J</b>) Dose-dependent inhibition of HCVcc JC1 virus infection of primary human hepatocytes (PHH). JC1 particles were pre-incubated with different doses of BLd-H77 (μg/ml) or with PBS and then used to infect PHH. PHH cell culture media were harvested four days post-infection and used to infect naïve Huh7.5 cells. Percentages of secondary infection are shown and calculated according to the viral titers of JC1 virus pre-incubated with PBS (mean ± SD; n = 3). (<b>K</b>) Dose-dependent inhibition of JC1-derived HCVpc (HCV primary cell culture-derived) of Huh7.5. HCVpc particles were pre-incubated with different doses of BLd-H77 (μg/ml) or with PBS and used to infect naïve Huh7.5 cells. Percentages of primary infection using HCVpc particles are shown and calculated according to the viral titers of JC1 virus pre-incubated with PBS (mean ± SD; n = 3). (<b>L</b>). Inhibition of HCV infection <i>in vivo</i> in humanized liver mice. JC1 infectious titers (FFU/ml) were obtained from two independent mice cohorts treated with 30μg (red, n = 3) or 150μg (blue, n = 2) of BLd-H77, or with PBS (black; cohort = 6) under a “prophylactic” protocol. BLd-H77 or PBS was injected one day prior virus infection with JC1 HCVcc particles and subsequent injections were performed at day 1, 7 and 14 post-infection. Sera were harvested 7 (Week 1), 14 (Week 2) and 21 days (Week 3) post-infection. HCV viral titers were determined through infection of Huh7.5 cells with mouse sera and quantification of FFU/ml. One non-engrafted liver mice and one non-infected mice were used as negative controls (green, n = 2). N.D., non-detected.</p

BIS analysis of dengue E coevolving networks during pre- and post-fusion steps.

<p>(<b>A</b>) Linear representation of Dengue E protein. Starting and ending residue positions of each E domain are indicated. E domains are annotated by distinct colors: DI, domain I (red); DII, domain II (yellow); DIII, domain III (blue); Tmd, transmembrane (black). (<b>B</b>) Organization and positions of the DENV E cluster 8 blocks (dark blue) on a mature E dimeric structure (PDB 1K4R). A linear representation of E is depicted and cluster block locations are indicated (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s004" target="_blank">S2 Table</a></b>). Cluster 8 coevolving residues located in areas where the two E monomers are in close contact are enlarged (green square). Structural proximity of two-coevolving cluster 8 internal loops located into two distinct Domain II (DII) sub-domains on the linear structure is also highlighted (red square). (<b>C</b>) Positions of E cluster 5 (red) and 10 (green) blocks on a mature tridimensional E monomer at a pre-fusion state (left, PDB 1K4R) and post-fusion state (right, PDB 1OK8). Stem and transmembrane domains are represented by a grey dotted line. A linear representation of E and cluster block locations are indicated for each cluster (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s004" target="_blank">S2 Table</a></b>). At the top of the panel, a schematic represents the current experimentally-validated fusion model of DENV, and how DENV E rearranges during this process. DENV E domains are colored into distinct colors (red, domain I; yellow, domain II; blue, domain III) as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g001" target="_blank">Fig 1A</a></b>.</p

BIS uncovers a coevolving signal between E1 and the Stem region that regulate HCV fusion.

<p>(<b>A</b>) Position of the three amino acid residues that differs between H77 (blue) and A40 (red) and are hypothesized to coevolve according to BIS prediction (gt1a cluster 5). The three H77 amino acids will be replaced by A40 residues individually or altogether to challenge BIS prediction. (<b>B</b>) Impact of the E1/Stem coevolution signal on HCV entry. Infectious titers of HCVpp viral particles harboring H77 (blue), A40 (red) and H77/A40 E1E2 chimera were determined. Two E1 H77 residues (S112, I117), a single H77 E2 residue (D462) or both (S112, I117, D462) were introduced into E1E2 A40. The different envelopes were incorporated at the surface of HCVpp, then used to infect Huh7.5. Infectious titers were quantified 72h post infection by flow cytometry (mean ± SD; n = 3). *<i>p</i><0.05, ns non-significant. (<b>C</b>) H77/A40 E1 and E2 chimera expression and incorporation onto HCVpp. Expression in transfected 293T cells (Cell lysates) and incorporation onto concentrated pseudoparticles (Viral Pellets) of E1 and E2 from the different H77/A40 chimera. Detection of E1 and E2 onto pseudoparticles harboring no envelope glycoproteins was used as negative control. MLV-Capsid (CA) was detected to control equivalent HCVpp production between chimera. (<b>D</b>). Impact of the E1/Stem coevolution signal on HCV fusion. LTRhiv-luciferase vector transduced 293T cells expressing the different E1E2 H77/A40 chimeric envelope glycoproteins were co-cultured with Tat-expressing Huh7.5 cells. Co-cultured cells were exposed to an acid shock (pH5, orange) or not (pH7, red) and luciferase activities were determined 72h post-exposure. Results are presented in relative light units (RLU) for each experimental condition (mean ± SD; n = 3). *<i>p</i><0.05, ***<i>p</i><0.001.</p

A dialog between E1 residues and the BL modulates virus entry and fusion.

<p>(<b>A</b>) Representation of the gt2 fusion cluster 5 (orange; dotted circle and linked by a bold line) as a putative mediator of E2 BL rearrangements and fusogenic conformational changes. Putative movements of the E2 BL are indicated by dotted double arrows. Rotation angles of the E2core structure and black cross are indicated as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g003" target="_blank">Fig 3B</a></b>. (<b>B</b>) Regions of interest to study the role of the cluster 5 (orange) and design of the gt2 chimera. Three regions were defined as cluster 5 blocks. One is located on E1 (30 aa; Region 1) and two are located in E2 BLd (Region 2: E2 408–436, N terminal region; Region 3: E2 437–456, C terminal region). (<b>C-D</b>) A dialog between E1 and the BL domain regulates virus entry. Infectious titers of HCVpp (GFP i.u./ml, <b>C</b>) and HCVcc (FFU/ml, <b>D</b>) viral particles harboring J6 (black), 2b1 (white) and J6/2b1 E1E2 chimera. Swapped regions (1, 2 or 3) are represented by white (2b1) or black (J6) boxes inserted into J6 or 2b1 linear representations, respectively (bottom). Infectious titers were quantified 72h (HCVpp) or 4 days (HCVcc) post infection of Huh7.5. (mean ± SD; n = 4); *<i>p</i><0.05.; ns non-significant. (<b>E-F</b>) A dialog between E1 and the BL domain regulates membrane fusion. Cell-cell fusion induced by the different E1E2 chimera (as described in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g005" target="_blank">Fig 5J</a></b>). HANA (Influenza Hemagglutinin-Neuraminidase) envelope glycoproteins were used as positive control. Data are presented as relative light unit (RLU) at pH5 (<b>E</b>) or as percentage of fusion (<b>F</b>) where pH7 RLU is considered as 100% fusion rate for each chimera (mean ± SD; n = 3); *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 ns non-significant. For panel (<b>F</b>), statistical significances were determined for each experimental condition versus control condition (100%).</p