Fig. 1: Reconstitution of functional A3H-Vif complex.

a Alignment of Vif sequences from various HIV-1/SIV strains. At amino acid position 48 (marked by a triangle), NL4-3 Vif has asparagine (N48), whereas LAI Vif has histidine (H48). b Alignment of primate A3H sequences. At amino acid position 97 (marked by a triangle), human A3H has lysine (K97), whereas chimpanzee A3H has glutamine (Q97). Conserved zinc-coordinating cysteines are marked by asterisks. hA3H, human; cpzA3H, chimpanzee; gA3H, gorilla; rmA3H, rhesus macaque; pgtA3H, pig-tailed macaque; gsnA3H, golden snub-nosed monkey; smA3H, sooty mangabey; agmA3G, African green monkey. c Vif-mediated degradation assay of A3H in HEK293T cells. A3H and Vif plasmids were co-transfected, and steady-state levels of A3H at post-48 h transfection were analyzed by Western blots. Quantified A3H levels are shown in the right-side chart. Assays were performed independently in triplicate (mean ± s.d.; n = 3). The statistical significance of A3H degradation was assessed by two-tailed t-test assuming equal variance; *P < 0.05; **P < 0.005; ***P < 0.0005; NS, not significant. P-values are 0.0477, <0.0001, <0.0001, and <0.0001 for WT A3H + WT Vif, WT A3H + N48H Vif, K97Q A3H + WT Vif, and K97Q A3H + N48H Vif, respectively. The addition of N48H mutation in Vif lowered the levels of both WT and K97Q A3H. K97Q mutation in A3H lowered the A3H levels when transfected with either WT or N48H Vif. An anti-α-tubulin antibody was used as a loading control. d Binding analysis of A3H and VCBCCR complex by SEC. The biggest peak shift was observed when K97Q A3H was combined with N48H NL4-3 Vif. The fractions corresponding to peak 1 were used for the cryo-EM study. MBP-fused A3H was used in this assay. SDS-PAGE gels show the protein components in the indicated SEC fractions. e In vitro ubiquitination assays. Vif-mediated ubiquitin chain initiation and elongation on the purified A3H were tested with or without ubiquitin-conjugating enzymes ARIH2/UBE2L3 and UBE2R1, respectively. Mono-ubiquitination and poly-ubiquitination were the most efficient when the K97Q A3H was combined with N48H NL4-3 Vif. Source data are provided as a Source Data file.