Figure 3.

Rotaviral nonstructural protein 1 triggers ubiquitination and proteasomal degradation of AGO2. (a) Whole cellular extracts from pcDNSP1‐ and pcDNSP4‐transfected MA104 cells were subjected to western blot analysis for checking expressions of AGO1, AGO2, Pan3, and Ser616 pDRP1. (b) MA104 cells were mock‐infected or infected with RV‐A5–13 and RV‐A5–16 (2–12 hpi). AGO2 and P53 expression was checked in cellular extracts of infected cells prepared at indicated time points. NSP1 protein expression was analysed in the same lysates to confirm RV‐A5–13 and RV‐A5–16 infection. (c) MA104 cells were kept untransfected or transfected with pcDNSP1; 6‐hr posttransfection, cells were treated with vehicle (DMSO) or 1‐μM Lactacystin. Cellular lysates prepared after 24 hr of transfection were subjected to SDS‐PAGE followed by immunoblotting and expression of AGO2 was checked. His protein expression was checked as a marker of pcDNSP1 transfection. (d) pcDNSP1‐transfected MA104 cells were treated with either vehicle (DMSO) or 500‐nM MLN4924 (6 hr after transfection). Whole‐cell lysates were prepared (24‐hr posttransfection), and immunoblotting was done to check expressions of AGO2, His (NSP1), Cullin1, and Cullin3. Neddylated form of Cullin1 and Cullin3 is indicated by arrows. (e) MA104 cells ectopically expressing pCMV‐FLAG‐Ub were cotransfected with either empty vector (pcDNA6B) or pcDNSP1. (Left panel) Cellular lysates prepared after 36 hr of transfection were immunoprecipitated with anti‐FLAG antibody, and immunoprecipitates were further subjected to immunoblotting with anti‐AGO2 antibody. Input lysates were probed with anti‐AGO2 and anti‐His (NSP1) antibodies. (Right panel) Reciprocal coimmunoprecipitation was done with anti‐AGO2 antibody followed by immunoblotting with anti‐FLAG antibody. Expressions of AGO2, His (NSP1) were checked in the input lysates. (f) MA104 cells were trasnfected with either empty vector (pcDNA6B), full‐length NSP1 (cloned in pcDNA6B vector), or truncated domains of NSP1 (RING and ΔRING domain cloned in pcDNA6B vector). After 36 hr of transfection, cell lysates were subjected to western blot analysis to check levels of AGO2 and P53. Expression of His tag was used as a marker of pcDNSP1, pcDRING‐NSP1, and pcDΔRING‐NSP1 transfection. (g) Equal amount of lysates from pCMV‐FLAG‐Ub overexpressed MA104 cells cotransfected with pcDRING NSP1 or pcDΔRINGNSP1 were immunoprecipitated with anti‐AGO2 antibody. Immunoprecipitates were then probed with anti‐FLAG antibody. Simultaneously, His (NSP1) and AGO2 expressions were assessed in the input lysates