Identification of Amino Acid Residues in APOBEC3G Required for Regulation by Human Immunodeficiency Virus Type 1 Vif and Virion Encapsidation

Abstract

The human immunodeficiency virus type-1 (HIV-1) accessory protein Vif serves to neutralize the human antiviral proteins apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G [A3G]) and A3F. As such, the therapeutic blockade of Vif function represents a logical objective for rational drug design. To facilitate such endeavors, we have employed molecular genetics to define features of A3G that are required for its interaction with Vif. Using alanine-scanning mutations and multiple different substitutions at key residues, we confirm the central role played by the aspartic acid at position 128 and identify proline 129 and aspartic acid 130 as important contributory residues. The overall negative charge of this 3-amino-acid motif appears critical for recognition by Vif, as single lysine substitutions are particularly deleterious and a double alanine substitution at positions 128 and 130 is far more inhibitory than single-residue mutations at either position. Our analyses also reveal that the immediately adjacent 4 amino acids, residues 124 to 127, are important for the packaging of A3G into HIV-1 particles. Most important are tyrosine 124 and tryptophan 127, and mutations at these positions can ablate virion incorporation, as well as the capacity to inhibit virus infection. Thus, while pharmacologic agents that target the acidic motif at residues 128 to 130 have the potential to rescue A3G expression by occluding recognition by Vif, care will have to be taken not to perturb the contributions of the neighboring 124-to-127 region to packaging if such agents are to have therapeutic benefit by promoting A3G incorporation into progeny virions.Proteins of the vertebrate apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) family protect cells against invasion by a broad range of viruses and mobile genetic elements, such as retroviruses, hepadnaviruses, endogenous retroviruses, and retrotransposons (3,5,8,16,30,42,46,47,52,56). These proteins belong to a larger family of proteins that contain one or two copies of the signature histidine/cysteine-X-glutamic acid-X 23-28 -proline-cysteine-X 2 -cysteine motif that is characteristic of cytidine and adenosine deaminases found in species ranging from bacteria to vertebrates (7,20,22,47). Expression of APOBEC3 proteins can lead to their encapsidation into progeny virions through recruitment to substrate viral or transposon capsid structures, and this appears to involve interactions with both Gag (or Gag-like) proteins and RNA (6,12,14,28,43,54,63).Following reverse transcription of the retroelement genomic RNA into single-stranded DNA, the APOBEC proteins can deaminate cytidines to uridines, causing deleterious mutations that result in the loss of genetic integrity and protein function, a process that is commonly referred to as hypermutation (19,30,64). More recently, it has emerged that hypermutation is not the only means by which APOBEC3 proteins can inhibit infectivity or transposition. Specifically, it has been reported that inhib...

HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain

“…In contrast to the residues that are involved in ubiquitination of A3G, amino acids implicated in Vif binding are located in the NTD (43) within the 128 DPD 130 motif (31,43) that is embedded in the 126 FWDPDYQ 132 (25) sequence in a loop preceding helix 4, which protrudes out from the overall structure (Fig. S2).…”

“…As can be readily observed, the critical residues involved in ubiquitination and proteasomal degradation of A3G (i.e., Lys 297, 301, 303, and 334) cluster in one region of the molecule. This area is located at the opposite end of the known interaction region with Vif (D128, P129, and D130) (31). Interestingly, the critical Lys residues are also close to the C terminus of the polypeptide chain, with K334 located within approximately 13Å.…”

“…Furthermore, extensive site-directed mutagenesis revealed that the 128 DPD 130 motif of A3G, located near the first Zn cluster, is crucial for direct binding to HIV-1 Vif. It is of interest that this motif is just downstream of residues 124 YYFW 127 , which are involved in A3G's ability to bind nucleic acids (31).…”

APOBEC proteins and intrinsic resistance to HIV-1 infection

“…The binding of HIV-1 Vif to A3G is critically dependent on an AspPro-Asp motif at positions 128-130 in A3G (Huthoff & Malim 2007). Satisfyingly, the recognition of this key element as a Vif-binding site has helped provide an explanation for the aforementioned species specificity of HIV/SIV Vif function ( §2).…”

“…Specifically, amino acid substitutions at Tyr-124 or Trp-127 yield proteins that are severely debilitated for packaging into HIV-1 particles, and are therefore poor inhibitors of virus infection (Huthoff & Malim 2007;Wang et al 2007;Zhang et al 2007). More recent results indicate that these mutant proteins are also substantially deficient for RNA binding and are unable to form A3G-A3G dimers efficiently (H. Huthoff & M. H. Malim 2008, unpublished observations).…”