Biomedical subjects

Sylvie Trassard

Publications and source records attributed to Sylvie Trassard.

4 recordsLinked to original sources

PubMed2006

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly.

Hepatitis C virus (HCV) core protein, expressed with a Semliki Forest virus replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. This model was used to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. Several mutants were designed as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. These data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly.

Animals↗

PubMed2003

Hepatitis C virus-like particle budding: role of the core protein and importance of its Asp111.

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.

Animals↗

PubMed2002

Demonstration of a tissue factor pathway inhibitor 2 messenger RNA synthesis by pure villous cytotrophoblast cells isolated from term human placentas.

Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.

Adult↗

PubMed2002

Hepatitis C virus-like particle morphogenesis.

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

Animals↗