These results indicate that both A3H_HapII activity and the steady-state levels of A3H_HapII are decreased by exposure to Vif

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These results indicate that both A3H_HapII activity and the steady-state levels of A3H_HapII are decreased by exposure to Vif. To examine whether weaker antiviral activity of A3H_HapI is due to its reduced expression compared to that of A3H_HapII, we compared the antiviral activity, intracellular expression, and virion packaging of increasing amounts of A3H_HapI versus A3H_HapII. was critical for determining A3H sensitivity and binding to HIV-1 Vif. The apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3) protein family is composed of cytidine deaminases that are capable of editing nucleic acids, converting cytidines to uracils (2,12,15,18,26,27,31,45,82,91). Members of this family of host cell proteins (APOBEC3A, -B, -C, -DE, -F, -G, and -H) have been shown to have differential inhibitory effects on various retroviruses and retroelements that are mediated through cytidine deamination and other mechanisms (4,7,8,10,13,14,19,20,22,23,25,37-41,43,46,48,55,57-59,63,71,72,75,77,79,81,89,93,94,98). In the absence of the HIV-1 protein Vif, APOBEC3G (A3G), like other APOBEC3 proteins, can be packaged into newly formed HIV-1 virions. Upon infection of new target cells, the packaged A3G induces C-to-U modifications in the newly reverse-transcribed minus-strand viral DNA, resulting in hypermutation of the viral genome (30,40,48,78,90,94). Virion-packaged A3G has also been reported to suppress viral activity by inhibiting reverse transcription and integration and/or by inducing viral DNA degradation AL082D06 (3,5,28,35,43,50,54,56,69,70,88). HIV-1 Vif is able to neutralize the antiviral activity of multiple APOBEC3 proteins by hijacking the host’s ubiquitin-proteasome system (16,36,48,49,51,73,76,92). This viral protein assembles with the host Cullin5-ElonginB-ElonginC E3 ligase complex (92) and polyubiquitinates APOBEC3 proteins for subsequent proteasome-mediated degradation (16,42,49,51,73,76,92). Mutational studies have shown that Vif interacts with ElonginB-ElonginC and Cullin5 through the S144LQxLA149and H108x5Cx17-18Cx3-5H139motifs located in the C terminus of Vif (44,51,52,83-85,92,93). HIV-1 Vif may also inhibit A3G function through degradation-independent mechanisms (60). Various motifs in the amino-terminal domain of HIV-1 Vif are responsible for its specific targeting of different APOBEC3 proteins (9,32,49,53,64,68,74,80). For example, several positively charged amino acids from position 22 to 44 are important for Vif-mediated suppression of A3G but not of A3F (9,21,53,64,74,87,96). In contrast, amino acids 11 to 17 and 74 to 79 of Vif are crucial for the Vif-mediated suppression of A3F but not A3G (32,64,68,74,80). In addition, a highly conserved hydrophobic motif, V52xIPLx4-5Lxx2YWxL72, in Vif is important for the suppression of both A3G and A3F (32,61). Other APOBEC3 family members, such as A3C and A3DE, are also subject to Vif-mediated degradation and are recognized by Vif through a mechanism similar to that for A3F (9,97). A3H lies distal to A3G on chromosome 22 (17,31,60), and it is the only APOBEC3 protein discovered thus far that contains a single copy of a Z3-type APOBEC3 catalytic domain (40) previously termed Z2 type (17). It encodes a conserved cytidine deaminase and is capable of inducing mutation in prokaryotes (19,29,58,59). A3H mRNA has been detected in several human tissues, including peripheral blood mononuclear cells, and is moderately upregulated in macrophages by interferons (29,58,59,79). When transfected into mammalian cells, it is insufficiently expressed and has weak antiretroviral activity (59). However, when a higher level of A3H expression in mammalian cells was achieved by using a cytomegalovirus (CMV) intron A-containing expression vector, A3H was found to have strong anti-HIV-1 activity (19,29). Several single-nucleotide polymorphisms (SNPs) were identified in the A3H gene. As first reported by OhAinle and colleagues (58,59), the Single Nucleotide Polymorphism database at NCBI (www.ncbi.nlm.nih.gov/projects/SNP) shows that there are six nonsynonymous SNPs (R18L, G37H, G105R, K121E/D, S140G, and E178D) AL082D06 and two single-codon deletions (14N and 15N) for the human A3H gene. Four haplotypes were reported to be circulating in the human population (29,58,95), as follows: HapI, 18R/105G/121K/178E; HapII, 18R/105R/121D/E/178D; HapIII, d15N/18R/105R/121D/E/178D; and HapIV, d15N/18L/105R/121D/E/178D. APOBEC3H variantNM_181773(Ensembl no. ENS 00000100298) has served as a wild-type reference and was designated HapI (58). Intriguingly, A3H_HapII, differing by only three amino acids from A3H_HapI (G105R/K121D/E/E178D), is more stable than the other three haplotypes and is reported to have the strongest inhibitory effect on both HIV-1 and Line-1 transposition (19,29,58,95). However, whether A3H can be suppressed by HIV-1 Vif remains controversial. While it has been argued that both HapI and HapII A3H have strong anti-HIV-1 activity AL082D06 and are insensitive to Vif degradation (19,29), OhAinle et al. have reported that A3H_HapII Rabbit Polyclonal to YOD1 is the only A3H variant with strong antiviral activity whose anti-HIV-1 activity is significantly decreased in the presence of AL082D06 Vif (58). It is also unknown whether HIV-1 Vif suppresses A3H_HapII by ubiquitin-proteasome mediated degradation, as is true for A3G. In the present study, we demonstrate that HIV-1 Vif does indeed interact with A3H_HapII and induces proteasome-mediated degradation through a mechanism similar to that responsible for A3G degradation. We have also found that.