The antiretroviral activity of the cellular enzyme APOBEC3G has been attributed to the excessive deamination of cytidine (C) to uridine (U) in minus strand reverse transcripts, a process resulting in guanosine (G) to adenosine (A) hypermutation of plus strand DNAs. The HIV-1 Vif protein counteracts APOBEC3G by inducing proteasomal degradation and exclusion from virions through recruitment of a cullin5 ECS E3 ubiquitin ligase complex. APOBEC3G belongs to the APOBEC protein family, members of which possess consensus (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C cytidine deaminase motifs. Earlier analyses of APOBEC-1 have defined specific residues that are important for zinc coordination, proton transfer, and, therefore, catalysis within this motif. Because APOBEC3G contains two such motifs, we used site-directed mutagenesis of conserved residues to assess each region's contribution to anti-HIV-1 activity. Surprisingly, whereas either the N- or C-terminal domain could confer antiviral function in tissue culture-based infectivity assays, only an intact C-terminal motif was essential for DNA mutator activity. These findings reveal the nonequivalency of APOBEC3G's N- and C-terminal domains and imply that APOBEC3G-mediated DNA editing may not always be necessary for antiviral activity. Accordingly, we propose that APOBEC3G can achieve an anti-HIV-1 effect through an undescribed mechanism that is distinct from cytidine deamination.

No abstract

To form an immature HIV-1 capsid, 1,500 HIV-1 Gag (p55) polypeptides must assemble properly along the host cell plasma membrane. Insect cells and many higher eukaryotic cell types support efficient capsid assembly, but yeast and murine cells do not, indicating that host machinery is required for immature HIV-1 capsid formation. Additionally, in a cell-free system that reconstitutes HIV-1 capsid formation, post-translational assembly events require ATP and a subcellular fraction, suggesting a requirement for a cellular ATP-binding protein. Here we identify such a protein (HP68), described previously as an RNase L inhibitor, and demonstrate that it associates post-translationally with HIV-1 Gag in a cell-free system and human T cells infected with HIV-1. Using a dominant negative mutant of HP68 in mammalian cells and depletion-reconstitution experiments in the cell-free system, we demonstrate that HP68 is essential for post-translational events in immature HIV-1 capsid assembly. Furthermore, in cells the HP68-Gag complex is associated with HIV-1 Vif, which is involved in virion morphogenesis and infectivity. These findings support a critical role for HP68 in post-translational events of HIV-1 assembly and reveal a previously unappreciated dimension of host-viral interaction.

To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of immature HIV capsids de novo in a cell-free system. Capsid authenticity is established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating the fidelity of the cell-free reaction. Assembly is separated into co- and posttranslational phases, and three independent posttranslational requirements are demonstrated: (a) ATP, (b) a detergent-sensitive host factor, and (c) a detergent-insensitive host subcellular fraction that can be depleted and reconstituted. Assembly appears to proceed by way of multiple intermediates whose conversion to completed capsids can be blocked by either ATP depletion or treatment with nondenaturing detergent. Specific subsets of these intermediates accumulate upon expression of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assembly. Furthermore, the accumulation of complexes of similar sizes in cells expressing the corresponding mutants suggests that comparable intermediates may exist in vivo. From these data, we propose a multi-step pathway for the biogenesis of HIV capsids, in which the assembly process can be disrupted at a number of discrete points.

The RNA helicase DDX6 promotes HIV-1 assembly in a co-opted cellular complex containing P body proteins and ABCE1.