Pierre-Yves Sizaret

Human papillomavirus types 16, 31, and 58 use different endocytosis pathways to enter cells.

ABSTRACT The early steps of the intracellular trafficking of human papillomavirus type 16 (HPV-16), -31, and -58 pseudovirions were studied by investigating the effects of drugs acting at defined points of endocytosis pathways on virus-like particle-mediated pseudoinfection by overexpression of a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis and by electron microscopy. The results obtained suggested the involvement of clathrin-mediated endocytosis in HPV-16 and HPV-58 entry and caveola-mediated endocytosis in HPV-31 entry.

Generation of Merkel Cell Polyomavirus (MCV)-Like Particles and Their Application to Detection of MCV Antibodies

ABSTRACT The genome of a new human polyomavirus, known as Merkel cell polyomavirus (MCV), has recently been reported to be integrated within the cellular DNA of Merkel cell carcinoma (MCC), a rare human skin cancer. To investigate MCV seroprevalence in the general population, we expressed three different MCV VP1 in insect cells using recombinant baculoviruses. Viruslike particles (VLPs) were obtained with only one of the three VP1 genes. High-titer antibodies against VP1 VLPs were detected in mice immunized with MCV VLPs, and limited cross-reactivity was observed with BK polyomavirus (BKV) and lymphotropic polyomavirus (LPV). MCV antibodies were detected in 77% of the general population, with no variations according to age.

Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking

ABSTRACT Hepatitis B virus (HBV) is unusual in that its surface proteins (small [S], medium, and large [L]) are not only incorporated into the virion envelope but they also bud into empty subviral particles in great excess over virions. The morphogenesis of these subviral envelope particles remains unclear, but the S protein is essential and sufficient for budding. We show here that, in contrast to the presumed model, the HBV subviral particle formed by the S protein self-assembles into branched filaments in the lumen of the endoplasmic reticulum (ER). These long filaments are then folded and bridged for packing into crystal-like structures, which are then transported by ER-derived vesicles to the ER-Golgi intermediate compartment (ERGIC). Within the ERGIC, they are unpacked and relaxed, and their size and shape probably limits further progression through the secretory pathway. Such progression requires their conversion into spherical particles, which occurred spontaneously during the purification of these filaments by affinity chromatography. Small branched filaments are also formed by the L protein in the ER lumen, but these filaments are not packed into transport vesicles. They are transported less efficiently to the ERGIC, potentially accounting for the retention of the L protein within cells. These findings shed light on an important step in the HBV infectious cycle, as the intracellular accumulation of HBV subviral filaments may be directly linked to viral pathogenesis.

Saccharomyces cerevisiae modulates immune gene expressions and inhibits ETEC-mediated ERK1/2 and p38 signaling pathways in intestinal epithelial cells.

Background Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. ETEC infections cause pro-inflammatory responses in intestinal epithelial cells and subsequent diarrhea in pigs, leading to reduced growth rate and mortality. Administration of probiotics as feed additives displayed health benefits against intestinal infections. Saccharomyces cerevisiae (Sc) is non-commensal and non-pathogenic yeast used as probiotic in gastrointestinal diseases. However, the immuno-modulatory effects of Sc in differentiated porcine intestinal epithelial cells exposed to ETEC were not investigated. Methodology/Principal Findings We reported that the yeast Sc (strain CNCM I-3856) modulates transcript and protein expressions involved in inflammation, recruitment and activation of immune cells in differentiated porcine intestinal epithelial IPEC-1 cells. We demonstrated that viable Sc inhibits the ETEC-induced expression of pro-inflammatory transcripts (IL-6, IL-8, CCL20, CXCL2, CXCL10) and proteins (IL-6, IL-8). This inhibition was associated to a decrease of ERK1/2 and p38 MAPK phosphorylation, an agglutination of ETEC by Sc and an increase of the anti-inflammatory PPAR-γ nuclear receptor mRNA level. In addition, Sc up-regulates the mRNA levels of both IL-12p35 and CCL25. However, measurement of transepithelial electrical resistance displayed that Sc failed to maintain the barrier integrity in monolayer exposed to ETEC suggesting that Sc does not inhibit ETEC enterotoxin activity. Conclusions Sc (strain CNCM I-3856) displays multiple immuno-modulatory effects at the molecular level in IPEC-1 cells suggesting that Sc may influence intestinal inflammatory reaction.

Biological and molecular features of the relationships between Diadromus pulchellus ascovirus, a parasitoid hymenopteran wasp (Diadromus pulchellus) and its lepidopteran host, Acrolepiopsis assectella.

The Diadromus pulchellus ascovirus (DpAV) has been isolated from laboratory strains of Diadromus pulchellus and in natural wild populations collected from the Antibes locality (southern France). The DpAV genome was found in the cells of the head, thorax and abdomen of this hymenopteran wasp. DpAV virions are present in the female genitalia and are transmitted to the nymphal lepidopteran host, Acrolepiopsis assectella, at each oviposition of the female wasp. The presence of the DpAV genome in all Diadromus somatic cells suggests that it is inherited by vertical transmission. DpAV is amplified in the host tissues during the larval development of D. pulchellus in A. assectella. Cell lysis due to amplification of the virus does not prevent the development of the hymenopteran larva. Virus amplification appears to be slower in nymphs parasitized by D. pulchellus than in nymphs artificially infected with DpAV alone. Lysis of the nymphal cells due to viral replication seems to be synchronous with egg hatching and the development of the hymenopteran larva. The features of DpAV and its relationship with the parasitoid wasp D. pulchellus during its development are compared to those of the ichnoviruses.

Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp111

ABSTRACT In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.

Insertion of a foreign sequence on capsid surface loops of human papillomavirus type 16 virus-like particles reduces their capacity to induce neutralizing antibodies and delineates a conformational neutralizing epitope

The aims of this study were to generate chimeric human papillomavirus (HPV)-16 L1 virus-like particles (VLPs) in order to identify immunogenic domains and conformational neutralizing epitopes, and to characterize the regions where a foreign epitope could be introduced. We hypothesized that these regions could be on L1 protein loops since they are exposed on the surface of VLPs. The aims of this study were achieved by mutating HPV-16 L1 proteins. Six amino acids encoding for the epitope 78-83 (DPASRE) of the hepatitis B core (HBc) antigen were introduced within the different loops of the L1 protein at positions 56/57, 140/141, 179/180, 266/267, 283/284 or 352/353. All these chimeric L1 proteins were capable of self-assembly into VLPs. The antigenicity and immunogenicity of some of these VLPs were reduced compared to the levels observed with wild-type VLPs. All were nevertheless able to induce neutralizing antibodies. VLPs with insertion at position 266/267 induced lower levels of neutralizing antibodies, suggesting the involvement of residues situated on FG loop in L1 neutralizing epitopes. All the chimeric L1 proteins except the one with insertion at position 56/57 were also able to induce anti-HBc antibodies, thus suggesting exposure of the HBc epitope on the VLP surface. Taken together, our findings indicate the possibility of designing HPV-derived vectors that are less immunogenic and suggest positions for insertion of defined immune epitopes or cell ligands into L1 protein to be exposed on the surface of VLPs.

Membrane characterization using microscopic image analysis

Abstract Five organic ultrafiltration membranes, made of different materials (PES, PVDF and PAN), have been visualized by a field emission scanning electron microscope (FESEM). Obtained images have been treated by the processing and analysis program NIH image and parameters such as, porosity ( A k ), pore density ( N ), mean pore radius ( r p ) and pore size distribution have been quantified for each membrane. The mean pore size obtained from image analysis agrees well with rejection data of fluorescein isothiocyanate dextrans, using the Ferry’s law approximation. The membrane thickness Δ x has been also measured on images. Results allowed the comparison between the A k /Δ x values obtained from image analysis and the A k /Δ x values obtained by diffusion experiments. Results, combined with porosity and water permeability values, gave information on the material hydrophilicity.

Rck of Salmonella enterica, subspecies enterica serovar Enteritidis, mediates Zipper-like internalization

Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rck-coated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.

CCL5-enhanced human immature dendritic cell migration through the basement membrane in vitro depends on matrix metalloproteinase-9

The proinflammatory chemokine CC chemokine ligand 5 (CCL5) is a potent chemoattractant of immature dendritic cells (iDCs). It remains to be elucidated whether CCL5 may also enhance iDC migration through the basement membrane by affecting matrix metalloproteinase (MMP)‐9 secretion. In this study, iDCs were differentiated in vitro from human monocytes of healthy donors. Zymographic analysis of cellular membranes of nontreated iDCs revealed a basal secretion of the pro‐ and active MMP‐9, whereas only pro‐MMP‐9 was detected in conditioned media. Increasing concentrations of CCL5 significantly enhanced MMP‐9 secretion by iDCs, peaking at 100 ng/ml, which optimally increased iDC migration through a reconstituted basement membrane (Matrigel™) in vitro. The CCL5‐enhanced secretion of MMP‐9 occurred early (2 h) and was maintained at least for 10 h. A significant increase in MMP‐9 mRNA synthesis was detected by reverse transcriptase‐polymerase chain reaction, only at 6 h of CCL5 treatment, which suggests that the early effect of CCL5 (0–4 h) on MMP‐9 secretion was independent of mRNA synthesis, whereas the more delayed effect (6–10 h) could be mediated through an increase in MMP‐9 gene expression. In a Matrigel migration assay, the CCL5‐enhanced iDC migration was reduced significantly by specific inhibitors of MMP‐9, such as tissue inhibitor of metalloproteinase‐1 or an anti‐MMP‐9 antibody, which indicates that iDC migration through the basement membrane depends on MMP‐9. These results suggest that under inflammatory conditions, the chemokine CCL5 may enhance iDC migration through the basement membrane by rapidly increasing their MMP‐9 secretion.