Fig. 6: Polyubiquitination is required for SERINC5 anti-HIV-1 activity.
A WT and ∆Nef HIV-1 were produced from HEK293T cells in the presence of Ser5 and its lysine mutants. Virions were purified from culture supernatants via ultracentrifugation. Ser5 expression in cell lysate and virions were detected by WB. B Ser5 was expressed with Nef in HEK293T cells in the presence of ectopic KLHL20 and Cul3 expression and/or their KDs by CRISPR/Cas9. Ser5 expression was compared by WB. C ∆Nef HIV-1 was produced from HEK293T and Jurkat cells in the presence of Ser5 and its lysine mutants. After normalization by p24Gag ELISA, viral infectivity was analyzed after infection of HIV-1 luciferase reporter cell line TZM-bI. D ∆Nef HIV-1 was produced from HEK293T and Jurkat cells in the presence of SERINC5, ectopic KLHL20 and Cul3 expression, and/or their KDs by CRISPR/Cas9. Viral infectivity was analyzed similarly as in C). For C and D, results are presented as relative values, with the infectivity of viruses produced in the presence of a control vector (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two or three experiments. n = 3 (C) or n = 2 (D); One-way ANOVA Tukey test; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were repeated twice, and representative experiments are shown. Source data are provided as a Source Data file.
