The current study seeks to continue the work from the foundation laid in the first grant. In particular the work will seek to define further the relationship between dengue virus serotype and proteins involved in the binding and internalization of the dengue virus. The work will proceed along two major lines:
1. Identification of receptor elements for different serotypes
We have already identified one protein involved in the binding of dengue virus serotype 2 to liver cells (Jindadamrongwech et al., Archives of Virology, DOI: 10.1007/s00705-003-0263-x). Evidence from VOPBA suggests that different proteins may be primarily involved in binding different serotypes. Specifically, identification of dengue virus binding proteins in HepG2 was performed by VOPBA at three different viral binding temperatures (4oC, RT, and 37 oC). At 4oC binding, DEN-2 bound a protein band of approximately 78-80 kDa and weakly bound to 90, 98, and 102 kDa proteins (figure 1). DEN-3 and DEN-4 were not efficiently bound at 4oC. However, the efficiency of binding was improved at room temperature and 37oC in which the higher temperature showed higher binding efficiency, especially in DEN-4. DEN-3 and DEN-4 bound to proteins of approximately 90, 130, 182 kDa and 90, 130 kDa, respectively. DEN-2 still bound predominantly to the 78-80 kDa protein but some weakly bands were lost at room temperature binding. These bands will be further investigated. The binding proteins will be excised from the gels, subjected to mass spectroscopy finger print analysis and candidate proteins investigated by antibody inhibition experiments.
2. Studies on additional cell lines.
To characterize the relationship between dengue viruses and C6/36 cell line, with respect to two main areas:
-Kinetics of viral propagation, viral production and internalization
-Receptor identification via VOPBA (Virus Overlay Protein-Binding Assays)
