Antigenic and structural analysis of the NS1 glycoprotein of dengue virus

Bletchly, Cheryl. (2002). Antigenic and structural analysis of the NS1 glycoprotein of dengue virus PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Bletchly, Cheryl.
Thesis Title Antigenic and structural analysis of the NS1 glycoprotein of dengue virus
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Prof John Mackenzie
Total pages 199
Language eng
Subjects 270303 Virology
321010 Infectious Diseases
Formatted abstract The dengue virus protein NS1, is the first non-structural protein to be translated in the dengue virus genome. It is glycosylated, which is unusual for a viral non structural protein and exists as an oligomer in its native form. The function of NS1 is still largely unknown however there is evidence that it is involved in RNA replication. It is also capable of inducing an immune response in experimental animals, with antibodies to NS1 being able to confer passive protection against lethal virus challenge. The work that comprises this thesis sought to expand our understanding of NS1 by exploring the human immune response to dengue virus infection and the role of NS1 in pathogenesis, and to analyze in detail the antigenicity and structure of the protein by building on our existing data.

Both the human immune response to NS1 and the circulation of NS1 in the serum of dengue virus infected patients was examined. An antigen capture ELISA was first developed for the detection of dengue virus NS1 based on the selection of monoclonal and polyclonal antibodies as the probe and capture reagents respectively. The limit of detection for NS1 with the method developed was 4 ng / mL for dengue-2 virus infections and 15 ng / mL for other dengue serotypes detected with a cross - reactive monoclonal antibody (MAb) probe. Sera were obtained from dengue virus infected patients in Thailand and screened for the presence of NS1 and antibody to NS1. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections in addition to high NS1 antibody concentrations, suggests that NS1 may be a significant contributor to the formation of circulating immune complexes that are hypothesized to play an important role in the pathogenesis of severe dengue disease.

Although NS1 has been reported to be immunogenic, little detailed information exists regarding the antigenic epitopes on the native dimeric form of the protein. In this thesis, using a panel of anti - NS1 MAbs in a competitive ELISA, five major antigenic domains and several sub - domains were defined for the secreted form of NS1. Together with PEPSCAN data obtained previously in this laboratory, important protective and antigenic epitopes in the primary and tertiary sequence of NS1 were mapped. Using NS1 derived from both mammalian (Vero) and insect (mosquito) cells, and in both soluble and membrane - associated forms, it was found that the membrane-associated NS1 produced in Vero cells lacked one important antigenic and protective epitope. This may explain differences noted in protection of lethally challenged mice by the different forms of NS1. Partial enzymatic digestions of purified NS1 combined with N-terminal sequencing of the protein fragments obtained and an analysis of their reactivity with the panel of MAbs, provided further insights into the antigenic structure of NS1 at the amino acid sequence level. Detergent phase separation experiments carried out on these proteolytic fragments have identified that the hydrophobic character of NS1 resides in the N-terminal half of the molecule. All of this data has collectively enabled the construction of a structural model for NS1 that is presented in Chapter 4.

Progress was made toward identifying the parameters required for crystallization of dengue-2 NS1 with the expectation that a crystal structure would assist in defining the function of the protein. As the amount of NS1 secreted from dengue virus infected cells is not produced in sufficient quantities to purify for crystallography studies, NS1 was expressed as a recombinant protein in the baculovirus expression system. Pulse-chase experiments defined the optimum conditions for expression of recombinant secreted NS1 from Spodoptera frugiperda cells. Recombinant NS1 was immunoaffinty purified and found experimentally to resemble the authentic protein in terms of cellular localization, secretion, oligomerization, glycosylation and antigenicity (using the antigenic epitopes defined in Chapter 4). Cross-linking and electron microscopy studies provided support for the hexameric structure of both native and recombinant NS1. The isoelectric point of NS1 was determined to be 5.7 and concentrated protein (from 5 - 7 mg / mL) was used in crystallography trials. Both microcrystals and crystals were obtained under specific buffer conditions after ten weeks and nine months of incubation respectively. Unfortunately, these crystals did not diffract and are therefore not useful in collating structural information. However, the successful establishment of a purification regime for NS1 has been achieved and is an important step towards successful crystallization and ultimately structure resolution.
Keyword Dengue viruses
proteins
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