Figure 4

RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent. (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. MA104 cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.