Characterization of Chikungunya pseudotyped viruses: Identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in E1 glycoprotein

Abstract

Chikungunya virus (CHIKV) is an alphavirus responsible for a number of large outbreaks. Here we describe the efficient incorporation of CHIKV envelope glycoproteins into lentiviral and rhabdoviral particles. Vectors pseudotyped with CHIKV envelope proteins efficiently transduced many cell types from different species. However, hematopoietic cell types were either partially or completely refractory. A mutation in E1 (A226V) has been linked with expansion of tropism for mosquito species, although differences in in vitro infection of mosquito cell lines have not been noted. However, pseudovirion infectivity assays detected subtle differences in infection of mosquito cells, suggesting an explanation for the changes in mosquito tropism. The presence of C-type lectins increased CHIKV pseudotyped vector infectivity, but not infection of refractory cells, suggesting that they act as attachment factors rather than primary receptors. CHIKV pseudotypes will serve as an important tool for the study of neutralizing antibodies and the analysis of envelope glycoprotein functions.

FIG. 1

Chikungunya virus envelope protein expression. Western-blot were performed using a mix of human sera from CHIKV infected individuals to detect protein expression in pseudotype producer 293T cell lysates and concentrated HIV-pseudotypes bearing CHIKV wt and E1A226V mutant envelope glycoproteins and VSV G protein. The positions of molecular mass markers (kDa) are shown on the right.

FIG. 2

Effect of lysosomotropic agents on pseudotype transduction. Bafilomycin A inhibition of RD cell transduction by MLV-A env (triangles), CHIKV gps (squares) and VSV-G (dots) pseudotypes. Results are expressed as percentage of no drug and represent the means of duplicate wells. The data is representative of four experiments.

FIG. 3

Relative cell tropism of CHIKV pseudotypes. GFP expression was used for parallel titration of CHIKV and VSV-G pseudotypes in a panel of cell types. Relative CHIKV pseudotype titer as compared to VSV-G pseudotype titer in each cell type is shown. The dotted line indicates 100 % infectivity for VSV-G pseudotypes. Data represent the average of at least two independent experiments.

FIG. 4

Pseudotype infectivity for wt and A226V CHIKV envelopes in mammalian cells. Equal amounts (normalized by p24 ELISA) of pseudotypes bearing CHIKV envelope wt or 226V mutant were titrated in parallel in different cell types. GFP titer was determined by flow cytometry. Each pseudotype was tested in triplicate for each cell type and error bars represent standard deviations (SDs). Differences in viral titers were analyzed by pairwise t-tests. Diamond indicates P > 0.3.

FIG. 5

Pseudotype infectivity for CHIKV envelope variants in mosquito cells. VSV pseudotypes bearing CHIKV envelope wt (Black bars) or 226V mutant (Grey bars) were titrated in parallel in (A) Ae. albopictus cell lines C6/36 and C7-10 and RD cells. Four replicates were performed for each pseudotype. (B) Ae. aegypti cells and RD cells. Each pseudotype was tested in triplicate. GFP titer was determined by flow cytometry. Error bars represent standard deviations (SDs). Differences in viral titers were analyzed by pairwise t-tests. Diamond indicates P < 0.3. Dot indicates P< 0.05

FIG. 6

DC-SIGN and DC-SIGNR enhance infectivity of CHIKV. envelope glycoprotein-bearing pseudotypes in 293T-REx cell lines The data are presented as fold-enhancement of infection in lectin expressing 293T-REx cells compared to parental 293T-REx cells. Error bars indicated standard deviations (SDs) of triplicate values from a representative experiment. Similar results were obtained in three independent experiments.