Secretion of flaviviral non-structural protein NS1: from diagnosis to pathogenesis.

Flaviviruses are major arthropod-borne human pathogens responsible for life-threatening encephalitis, hepatitis and haemorrhagic fevers. These enveloped, single-stranded, positive-sense RNA viruses encode a polyprotein precursor of about 3400 amino acids, processed into three structural and seven non-structural proteins. The non-structural glycoprotein NS1 is essential for flavivirus viability. During host-cell infection in vitro, NS1 is found associated with intracellular organelles as a requisite for its role in viral replication, or is transported to the cell surface where it may trigger specific signalling pathways. In addition, a secreted form of the protein is released from flavivirus-infected mammalian cells. We have previously shown that the NS1 protein circulates during the acute phase of the disease in the plasma of patients infected with dengue virus type 1 and have extended our retrospective studies to dengue type 2 and type 3 cohorts, confirming the value of the NS1 antigen as an alternative diagnostic marker. Interestingly, detection of the NS1 protein in yellow fever virus and West Nile virus infections suggests that NS1 secretion is a hallmark of human flavivirus infections. The objectives of our current studies are to define the biological properties of the secreted form of the NS1 protein, to evaluate its possible contribution to viral pathogenesis, and to validate this protein as a candidate target for passive immunoprophylaxis against flaviviruses.

In vitro reconstructed mucosa-integrating Langerhans' cells.

All three-dimensional in vitro mucosal models constructed, thus far, have only been reconstituted by epithelial cells. We have developed a reconstructed oral and vaginal epithelium that integrates Langerhans' cells (LC), the dendritic cells (DC) of malpighian epithelia. The epithelium was composed of gingival or vaginal keratinocytes seeded on a de-epidermized dermis (DED) and grown in submerged culture for 2 weeks. LC precursors, obtained after differentiation of cord blood-derived CD34+ hematopoietic progenitor cells (CD34+HPC) by granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and Flt3-ligand (Flt3-L), were introduced after 6-8 days of culture into the reconstituted epithelium. The in vitro reconstituted mucosal epithelium formed a multilayered, well-differentiated epithelial structure, confirmed by the immunohistochemical expression of cytokeratins 4, 6, 10, 13, 14, 16 and involucrin. LC were identified in the basal and suprabasal epithelial layers by CD1a antigen, S100 protein and Langerin/CD207 expression, and by transmission electron microscopy. Type IV collagen was expressed at the chorio-epithelial junction, and most ultrastructural features of this junction were visualized by electron microscopy. This in vitro reconstructed gingiva or vagina integrating LC represents interesting models very similar to native tissues. Because LC play an important role in the mucosal immune system, our models could be useful for conducting studies on interactions with pathogenic agents (viruses, bacteria etc.), as well as in pharmacological, toxicological and clinical research.

Identification of three major phosphorylation sites within HIV-1 capsid. Role of phosphorylation during the early steps of infection.

We previously reported the presence of two cellular serine/threonine protein kinases incorporated in human immunodeficiency virus type 1 (HIV-1) particles. One protein kinase is MAPK ERK2 (mitogen-activated protein kinase), whereas the other one, a 53-kDa protein, still needs to be identified. Furthermore, we demonstrated that the capsid protein CAp24 is phosphorylated by one of those two virion-associated protein kinases (Cartier, C., Deckert, M., Grangeasse, C., Trauger, R., Jensen, F., Bernard, A., Cozzone, A., Desgranges, C., and Boyer, V. (1997) J. Virol. 71, 4832-4837). In this study, we showed that CAp24 is not a direct substrate of MAPK ERK2. Moreover, using site-directed mutagenesis of each of the 9 serine residues of CAp24, we demonstrated the phosphorylation of 3 serine residues (Ser-109, Ser-149, and Ser-178) in the CAp24. Substitution of each serine residue did not affect viral budding, nor viral structure. By contrast, substitution of Ser-109, Ser-149, or Ser-178 affects viral infectivity by preventing the reverse transcription process to be completely achieved. Our results suggest that CAp24 serine phosphorylation is essential for viral uncoating process.