Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway
Fig 5
NiV-C has a direct interaction with Tsg101.
(A) HA-tagged NiV-C co-immunoprecipitates endogenous Tsg101 from 293T cell lysates, as does HA-tagged Vps28, a known cellular interacting partner of Tsg101. (B) NiV-C was truncated as indicated, with all constructs retaining a C-terminal HA tag. (C) Loss of the CTD of NiV-C specifically leads to loss of pulldown of Tsg101. (D) A fusion of EGFP and NiV-C-CTD (HA-EGFP-C-CTD) pulls down Tsg101, whereas EGFP alone does not. (E) The C terminal domain (CTD) of Tsg101 was defined as in [31]. FLAG-Tsg101 was truncated as indicated. UEV, ubiquitin E2 variant domain; PRO, proline-rich domain; CC, coiled-coil region; and CTD, C-terminal domain. (F) Truncation of the CTD of Tsg101 results in loss of co-immunoprecipitation with NiV-C. (G) A fusion of EGFP and the full CTD (amino acids 303–390) of Tsg101 (FLAG-EGFP-Tsg101-CTD) co-immunoprecipitates with NiV-C, whereas EGFP alone does not. (H) Left panel, Coomassie-stained gel showing recombinant proteins (100 ng each lane) purified from E. coli as described in Materials and Methods. Right panel, Western analysis with repeat of Fig 5G except with purified recombinant proteins, showing that the interaction between NiV-C and Tsg101-CTD is direct.