Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation have not been elucidated. APOBEC3 family proteins play numerous functions in primates including humans. For instance, APOBEC3A initiates the mutations of foreign DNA (e.g., microbial DNA), which leads to the clearance of bacteria from human cells [7]. In addition, APOBEC3B-mediated mutation closely associates with several human cancers [8], [9], particularly breast cancer [10]. APOBEC3G is the most extensively analyzed APOBEC3 protein in the field Quizartinib of virology and plays a crucial role in the infection and replication of HIV-1, a causative agent of AIDS [11]. APOBEC3G is usually incorporated into HIV-1 particles and induces G-to-A mutations in the newly synthesized viral DNA, which results in the abrogation of viral replication [4], [12]. On the other hand, an HIV-1-encoded protein, viral infectivity factor (Vif), impedes APOBEC3G incorporation into progeny virions by degrading these proteins through the ubiquitin/proteasome-dependent pathway [4], [12]. In addition to APOBEC3G, studies using cell culture systems have exhibited that like APOBEC3G, APOBEC3F and APOBEC3D also potently impair HIV-1 replication [13]C[15]. However, one study has concluded that APOBEC3F expression levels in T cell lines were not sufficient to inhibit HIV-1 replication [16]. Another study analyzed the replication of HIV-1 Vif mutants that were defective in inducing degradation of APOBEC3G or APOBEC3F in main CD4+ T cells, and concluded that APOBEC3G exerts a stronger antiviral activity on HIV-1 than APOBEC3F [17]. Thus, the relative impact of different APOBEC3 proteins on HIV-1 replication has not been determined. Apart from their anti-HIV-1 abilities, certain studies have suggested that APOBEC3-mediated G-to-A mutation can lead Quizartinib to viral Quizartinib development and divergence [18]C[21]. However, it remains unclear how and which endogenous APOBEC3 proteins impact HIV-1 replication, pathogenesis, and diversity (NOG) mouse [22]C[28]. Our humanized mouse model is able to recapitulate the characteristics of HIV-1 pathogenesis such as the depletion of peripheral CD4+ T cells [22], [23], [25]. By using this model, we have previously demonstrated that this expression levels of endogenous APOBEC3 genes in human CD4+ T cells of humanized mice were comparable to those of humans and that the combined activity of endogenous APOBEC3 proteins can potently abrogate is not yet known. In fact, although G-to-A mutations, presumably caused by endogenous APOBEC3 proteins, have been clearly observed in the viral genomes of HIV-1-infected patients, the frequencies of G-to-A mutations seem to vary among individuals and the mutation context is still controversial [21], [29]C[37]. Moreover, because there is a possibility that some endogenous APOBEC3 protein(s) are capable of facilitating viral diversification mutants, which are unable to degrade APOBEC3D/F, APOBEC3G, or both APOBEC3D/F and APOBEC3G, are severely impaired, demonstrating that endogenous APOBEC3D/F and APOBEC3G proteins potently suppress HIV-1 propagation and thereby facilitate viral adaptation and development. Results Strong inhibition of HIV-1 propagation by mutating 14DRMR17 and/or 40YRHHY44 motifs in Vif It was exhibited that 14DRMR17 motif in Vif is necessary for the degradation of APOBEC3D and APOBEC3F, while 40YRHHY44 motif in Vif is necessary for the degradation of APOBEC3G [38], [39]. As shown in Physique 1A, these two motifs were highly conserved in HIV-1 group M. In addition, these motifs are located on the outside regions of Vif protein (Physique 1B) [39]. Moreover, we confirmed that APOBEC3D, APOBEC3F, and APOBEC3G have the ability to decrease (Physique S1). Physique 1 Anti-HIV-1 effect of APOBEC3 proteins mutants did not propagate efficiently (Figures S2A and S2B). p85 These findings strongly suggest Quizartinib that endogenous APOBEC3 proteins, particularly APOBEC3D, APOBEC3F and APOBEC3G, can potently impair HIV-1 propagation mutant contamination in humanized mice. To quantitatively analyze the magnitude of viral propagation and that the antiviral activity of endogenous APOBEC3G is usually higher than the combined antiviral activity of APOBEC3D and APOBEC3F. No reversion of mutations in HIV-1 mutants Even though growth of HIV-1 mutants was generally low, certain mice infected with 4A HIV-1 exhibited moderate levels of viremia (Physique 2A). To assess the possibility that reversion of mutations in led to the limited spread of HIV-1 mutants in humanized mice, we analyzed.