FIG 4.

Hsp90β is required for CHIKV replication and interacts with nsP2 protein. Gene silencing of Hsp90 family proteins, Hsp90α, Hsp90β, Grp94, and TRAP1. HEK293T cells were transfected with 100 nM nontargeting (NT) siRNA or siRNA Hsp90α, Hsp90β, Grp94, or TRAP1. At 48 h posttransfection, cells were infected with CHIKV at an MOI of 1. (A and B) After 12 hpi, cell lysates were collected and subjected to (A) Western blot and (B) RT-qPCR analyses. Statistical analyses were determined using paired Student’s t tests, and significant differences are indicated by * (P < 0.05). (C) Immunoprecipitation assay of GFP-tagged CHIKV-nonstructural proteins with Hsp90β. Briefly, GFP only (empty^GFP) and GFP-tagged CHIKV nsPs were expressed in HEK-293T cells for 24 h. At 24 h posttransfection, cell lysates were subjected to coimmunoprecipitation using 1 μg of anti-GFP antibodies. Western blot analysis was performed to determine the interaction between CHIKV nsPs and Hsp90β. Isotype control IgG was used as a negative control to pull down the protein complex.