Biochemical Fractionation and Purification of High-Molecular-Mass APOBEC3G Complexes
Human APOBEC3G (A3G) is a cytidine deaminase that broadly restricts the replication of many retroviruses, including HIV-1. In different cell types, cytoplasmic A3G is expressed in high-molecular-mass (HMM) RNA–protein complexes or low-molecular-mass (LMM) forms displaying different biological activities. LMM A3G has been proposed to restrict HIV-1 infection soon after virion entry in resting CD4 T cells, monocytes, and mature dendritic cells. Cellular activation and specific cytokine signaling promote the recruitment of LMM A3G into HMM complexes that are likely nucleated by the induced expression of Alu retroelement RNAs. HMM A3G sequesters these retroelement RNAs away from the nuclear LINE-derived enzymes required for Alu retrotransposition. However, assembly of A3G into HMM complexes suppresses its enzymatic activity and may render cells permissive to HIV-1 infection. During HIV-1 virion formation, newly synthesized LMM A3G is preferentially encapsidated when the HIV-1 viral protein viral infectivity factor is absent and employs sequential actions to restrict HIV-1. A3G’s biological activities are tightly regulated by its ability to assemble into HMM complexes. Here, we describe in detail the procedures for biochemical fractionation and purification of HMM A3G complexes. Purified HMM A3G complexes will be useful for studying many aspects of the A3G biology, including A3G’s roles in restricting retroviral replication, inhibiting retroelement mobility, and potentially regulating cellular RNA function.
Key wordsAPOBEC3G Cytidine deaminases HIV-1 Vif Alu Retrotransposition FPLC Tandem affinity purification High-molecular-mass Low-molecular-mass
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