Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

Abstract

The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

Figure 1

Generation of an E2 transposon insertion library. A schematic outlining the strategy employed to generate the library of E2 transposon mutants.

Figure 2

Distribution of transposon insertions in E2. A: A schematic of the SINV E2 protein. The hatched area represents the transmembrane (TM) region. The glycosylation sites at 196 and 318 are represented by a stick and ball. CT: cytoplasmic tail. B: location of all identified transposon insertions. The insertion sites are represented as bars with the residue after which the insertion occurred indicated by arrows at the top or bottom. C: location of possible hotspots for transposition (bars). The number of independent clones with insertions at that particular location is show in parentheses. The gray shaded area represents the putative receptor binding region where no transposon insertions were recovered. D: distribution of transposon insertions resulting in a lethal phenotype; E: large plaque phenotype (wild type); F: medium plaque phenotype and G: small plaque phenotype.

Figure 3

One-step growth curves of selected transposon mutants. The growth kinetics of the transposon mutants was compared to wild type SINV in BHK cells. A: transposon mutants with large plaque phenotype. B: transposon mutants with medium plaque phenotype. C: transposon mutants with small plaque phenotype. Media was replaced every 30 minutes for the first two hours, and then every hour for 12 hours post-infection. Supernatant was collected at 4, 6, 8, 10 and 12 hours post-infection and released virus was assayed by titration on BHK cells at 37ºC. The results represent the average of two experiments.

Figure 4

Surface expression of E2. BHK cells were infected with wild-type SINV (A) or an E2 transposon insertion mutant (E2-53) (B) at a multiplicity of infection of 1. Cells were harvested 12 hours post-infection and stained with a polyclonal α-E2 antibody and a fluorescein-conjugated secondary antibody. Cells were analyzed using a FACSCalibure flow cytometer. The grey filled-in curve represents the signal from uninfected BHK cells. The shift in the signal upon virus infection is shown by the clear curve and represents the expression of E2 at the cell surface. The number above the curve is the geometric mean for the region of the curve delineated by the line and is a measure of the amount of E2 at the plasma membrane. The results represent data from a single experiment. However, the experiment has been repeated three times, with consistent results.

Figure 5

Neutralization of E2 transposon insertion mutations by MAb 202 and MAb 209. 100 PFU of Toto64 or E2-119 were each incubated with different concentrations of MAb 202 (A) or MAb 209 (B) for 30 minutes. The titer of the resulting virus was determined by standard plaque assay on BHK cells at 37ºC. The results are the average of three experiments. The error bars indicate the standard deviation.

Figure 6

Functional regions of SINV E2. The functional regions of E2 based on the characterization of the E2 transposon linker-insertion library have been mapped onto a schematic of SINV E2. The color scheme represents the plaque phenotype of the insertion mutants. Blue represents a wild type-like phenotype (large plaque), green represents a medium plaque phenotype, brown represents a small plaque phenotype and red indicates that the insertions were lethal.