Functional characterization of the alphavirus *E2 glycoprotein: A guide to host range modification

Chanakha K Navaratnarajah, Purdue University

Abstract

The cell receptor-binding and host cell membrane fusion activities of the alphavirus, Sindbis virus (SINV), are performed by two distinct envelope glycoproteins, E2 and E1 respectively. This separation of function suggests that the cell receptor binding activity of E2 could be modified with minimal effect on the cell membrane fusion activity of E1, thus suggesting the use of SINV for developing targeted viral vectors. Based on structural data, we have produced a recombinant SINV expressing the human epidermal growth factor (hEGF) at the N-terminus of E2. The hEGF-targeted virus displayed enhanced infection of hEGF receptor-positive cells. In order to restrict the natural tropism of the virus, alanine scanning mutations were made in a region of E2 glycoprotein (amino-acids 170-220) shown to be important for receptor binding. These experiments revealed several mutations that may impair the virus-receptor interaction, and thus, help to minimize the natural tropism of the virus. A transposon-based linker insertion mutagenesis system was used to create a library of 19-amino-acid insertions in the SINV E2 glycoprotein. These insertion mutations were characterized in terms of protein expression, transport of E2 to the plasma membrane and viability. A majority of the insertions in E2 were tolerated and gave rise to viable virus. Insertions that resulted in a wild-type like phenotype were especially interesting as they represented sites that could be used to insert targeting-ligands with minimal effects on virus growth. Several insertions that may have impaired virus-receptor interactions were also identified. In addition to the molecular genetic characterization of E2, expression and purification of different C-terminal truncations of the glycoprotein was attempted as a first step towards the structural characterization of the protein. Two truncations of Ross River virus (RRV) E2 (E2-193 and E2-250) were successfully expressed by a baculovirus insect cell expression system. Although the purified proteins exhibited a similar antigenicity as the native full-length E2 protein, the pattern of glycosylation was shown to be different.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Molecular biology|Microbiology|Virology

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