The human genome encodes seven APOBEC3 (A3) cytidine deaminases with potential antiretroviral activity: A3A, A3B, A3C, A3DE, A3F, A3G, and A3H. A3G was the first identified to block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3F, A3B, and A3DE were shown later to have similar activities. HIV-1 produces a protein called Vif that is able to neutralize the antiretroviral activities of A3DE, A3F, and A3G, but not A3B. Only the antiretroviral activity of A3H remains to be defined due to its poor expression in cell culture. Here, we studied the mechanism impairing A3H expression. When primate A3H sequences were compared, a premature termination codon was identified on the fifth exon of the human and chimpanzee A3H genes, which significantly decreased their protein expression. It causes a 29-residue deletion from the C terminus, and this truncation did not reduce human A3H protein stability. However, the mRNA levels of the truncated gene were significantly decreased. Human A3H protein expression could be restored to a normal level either by repairing this truncation or through expression from a vector containing an intron from human cytomegalovirus. Once expression was optimized, human A3H could reduce HIV-1 infectivity up to 150-fold. Importantly, HIV-1 Vif failed to neutralize A3H activity. Nevertheless, extensive sequence analysis could not detect any significant levels of G-to-A mutation in the HIV-1 genome by human A3H. Thus, A3H inhibits HIV-1 replication potently by a cytidine deamination-independent mechanism, and optimizing A3H expression in vivo should represent a novel therapeutic strategy for HIV-1 treatment.

Although the covalent attachment of a polyubiquitin is the prevailing paradigm for entry into proteasomes, accumulating evidence suggests that poorly defined ubiquitin-free pathways also degrade proteins. The cytidine deaminase APOBEC3G (A3G) potently inhibits human immunodeficiency virus type 1 replication by disrupting viral reverse transcription. However, human immunodeficiency virus type 1 produces a viral infectivity factor (Vif) to destroy this antiretroviral protein. It was shown that Vif binds to both A3G and a Cullin 5 ubiquitin-protein isopeptide ligase. It is currently accepted that this enzyme polyubiquitylates A3G on lysine residues, resulting in its degradation by proteasomes. Here, we find that A3G without ubiquitylation is still degraded by proteasomes in a Vif-dependent manner. We further show that Vif is polyubiquitylated and that this event could be critical for A3G proteasomal degradation. Thus, A3G is degraded by a novel pathway that might involve ubiquitylation of one protein and then targets a second binding partner for proteasomal entry and degradation. We propose that instead of triggering A3G polyubiquitylation, polyubiquitylated Vif might serve as a vehicle to transport A3G into proteasomes for degradation.Eukaryotes employ proteasomes to destroy misfolded proteins and control levels of protein expression. The 26 S proteasome, composed of a 20 S proteolytic cylinder and two 19 S regulatory caps, specializes in recognition and proteolysis of proteins covalently derivatized with a polyubiquitin chain on certain lysine residues (1). The process of polyubiquitylation involves three enzymes: E1, 2 E2, and E3 (2). E1 activates ubiquitin for transfer to E2. E2 interacts with a specific E3 partner, a multiprotein complex that binds directly to the substrate protein, to ensure the target specificity of ubiquitin conjugation. After a single ubiquitin is attached, additional ubiquitins can be linked to one of the seven lysines of ubiquitin to form a multiubiquitin chain, which serves as a signal for proteasomal degradation. Although this model has been verified for a great number of proteins, it also has been reported that some proteins can be degraded in proteasomes in the absence of polyubiquitylation (reviewed in Refs. 3 and 4).On human chromosome 22, a cluster of genes encodes a family of seven APOBEC3 (apolipoprotein B mRNA-editing catalytic polypeptide 3; A3) antiretroviral proteins: A3A, A3B, A3C, A3DE, A3F, A3G, and A3H. They are cytidine deaminases with one or two cytidine deaminase domains (reviewed in Ref. 5). A3G potently blocks HIV-1 replication (6). However, HIV-1 produces a protein called Vif to counteract A3G by triggering its proteasomal degradation (7-9). Vif was shown to bridge A3G with a cellular E3 ligase (10). In fact, Vif has a BC-box motif (Ser 144 /Leu 145 /Gln 146 ) that binds to elongin C (EloC) (11, 12) and an HCCH motif (Cys 114 /Cys 133 ) that binds to Cullin 5 (Cul5), the core subunits of a Cul5-based E3 ligase (13-15). As a consequence, it is believed that A3G is p...