Identification of a region on the G2 protein of La Crosse encephalitis virus that is required for binding of the G2 protein to the surface of mosquito midguts Identification of a region on the G2 protein of La Crosse encephalitis virus that is required for binding of the G2 protein to the surface of mosquito midguts. Li-lin Cheng, Thomas M. Yuill and Barbara A. Israel. In order to examine the binding of G2 to mosquito midguts in more detail, we cloned the G2 gene into a mammalian cell expression plasmid, under the control of the T7 polymerase promoter. We generated six mutations in the G2 gene by replacing specific amino acid regions with the comparable sequence from Bunyamwera virus and we have expressed these six mutant proteins in HeLa cells.
All mutations result in the expression of protein on the surface of the HeLa cells.
In order to determine which regions of G2 are critical for binding to mosquito midgut cells, we tested the ability of HeLa cells infected with LACV, or transfected HeLa cells expressing native G2 or the mutated G2 proteins to bind to the lumen of isolated mosquito midguts. The mosquito midguts were opened to expose the lumen and were affixed to glass slides. HeLa cells were incubated on the midguts. The slides were gently washed, and the bound HeLa cells were detected by binding of anti-G2 Mabs to the surface of the cells. Six regions of the G2 protein were replaced, using in vitro mutagenesis, by regions of the Bunyamwera virus G2 protein.