Abstract
BackgroundHost restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP) from its N- or C-terminal fragments.ResultsThe results obtained with catalytic domain 1 and 2 (CD1 and CD2) mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22) to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity.ConclusionThese studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules.
Highlights
Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation
A3G-N-terminal 1–172 amino acids of YFP (NY) and A3G-C-terminal 173–238 amino acids of YFP (CY) express A3G that was fused to the NY or CY fragments of yellow fluorescent protein (YFP) at the C-terminus, respectively; A3G and the YFP fragments are separated by a 12 amino acid glycine-rich flexible linker (PGISGGGGGILD)
The results showed that all bimolecular fluorescence complementation assay (BiFC) fusion proteins were expressed; the A3G-NY and NY-A3G proteins were expressed at lower levels than the A3G-CY and CY-A3G proteins
Summary
Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Human immunodeficiency virus type 1 (HIV-1) has infected over 33 million people in the world, leading to the AIDS pandemic http://www.who.int. Recent discovery of intracellular host restriction factors suggests that HIV-1 must overcome these defenses in order to replicate and cause AIDS [1,2]. A3G, a member of the APOBEC3 family of proteins, is a host restriction factor that potently inhibits the replication of HIV-1 vectors that fail to express a functional Vif protein [1]. HIV-1 expresses the Vif protein, which binds to A3G and targets it for proteasomal degradation [9,10,11,12,13,14]
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