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APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

Sato K, Takeuchi JS, Misawa N, Izumi T, Kobayashi T, Kimura Y, Iwami S, Takaori-Kondo A, Hu WS, Aihara K, Ito M, An DS, Pathak VK, Koyanagi Y - PLoS Pathog. (2014)

Bottom Line: Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo.We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F.Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, Kyoto, Kyoto, Japan.

ABSTRACT
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 in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

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Expression levels of APOBEC3 and IFNB in infected humanized mice.(A) Dispersion of HIV-1 growth efficiency in humanized mice. (Left) The peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) were classified into 4 degrees (less than 103, 103–104, 104–105, or more than 105), and the distribution is plotted. (Right) The coefficient of variance of the peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) is shown. (B) Expression levels of APOBEC3D, APOBEC3F, and APOBEC3G in the splenic human CD4+ T cells of humanized mice (n = 73) were analyzed by real-time RT-PCR. The values were standardized as previously described [43], and the level of APOBEC3D is set to 1 to facilitate comparison. (C) Expression levels of APOBEC3D, APOBEC3F, APOBEC3G, and IFNB in the splenic human CD4+ T cells of infected mice (WT, n = 13; 4A, n = 20; 5A, n = 17; and 4A5A, n = 15) and mock-infected mice (n = 8) at 6 wpi were analyzed by real-time RT-PCR. Horizontal bars represent the averages. Asterisks represent statistically significant difference (P<0.05 by Student's t test) between infected mice and mock-infected mice. (D) Negative correlation between VL and APOBEC3 expression in 4A HIV-1-infected humanized mice. The mRNA expression levels of APOBEC3D (left), APOBEC3F (middle), and APOBEC3G (right) in the splenic human CD4+ T cells (x-axes) and the VL at 6 wpi (y-axis) of 4A HIV-1-infected mice (n = 20) are shown. The lines represent exponential approximation. Pearson correlation coefficient (r) was adopted to determine statistically significant correlation between each value.
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ppat-1004453-g003: Expression levels of APOBEC3 and IFNB in infected humanized mice.(A) Dispersion of HIV-1 growth efficiency in humanized mice. (Left) The peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) were classified into 4 degrees (less than 103, 103–104, 104–105, or more than 105), and the distribution is plotted. (Right) The coefficient of variance of the peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) is shown. (B) Expression levels of APOBEC3D, APOBEC3F, and APOBEC3G in the splenic human CD4+ T cells of humanized mice (n = 73) were analyzed by real-time RT-PCR. The values were standardized as previously described [43], and the level of APOBEC3D is set to 1 to facilitate comparison. (C) Expression levels of APOBEC3D, APOBEC3F, APOBEC3G, and IFNB in the splenic human CD4+ T cells of infected mice (WT, n = 13; 4A, n = 20; 5A, n = 17; and 4A5A, n = 15) and mock-infected mice (n = 8) at 6 wpi were analyzed by real-time RT-PCR. Horizontal bars represent the averages. Asterisks represent statistically significant difference (P<0.05 by Student's t test) between infected mice and mock-infected mice. (D) Negative correlation between VL and APOBEC3 expression in 4A HIV-1-infected humanized mice. The mRNA expression levels of APOBEC3D (left), APOBEC3F (middle), and APOBEC3G (right) in the splenic human CD4+ T cells (x-axes) and the VL at 6 wpi (y-axis) of 4A HIV-1-infected mice (n = 20) are shown. The lines represent exponential approximation. Pearson correlation coefficient (r) was adopted to determine statistically significant correlation between each value.

Mentions: In the 3 kinds of HIV-1 vif mutants, it was noteworthy that the VL in each 4A HIV-1-infected mouse varied between individual mice, while those in 5A and 4A5A HIV-1-infected mice were uniformly low (Figure 3A, left panel). In fact, the coefficient of variance of peak VL, which indicates the extent of distribution, in 4A HIV-1-infected mice was ∼2-fold higher than that in WT, 5A, and 4A5A HIV-1-infected mice (Figure 3A, right panel). These findings raised a possibility that the level of viremia in 4A HIV-1-infected mice is correlated with endogenous APOBEC3 expression levels. To address this possibility, we determined the endogenous expression levels of APOBEC3D, APOBEC3F, and APOBEC3G in the spleen of humanized mice by real-time RT-PCR and standardized them to those of APOBEC3D according to the procedure reported previously [42], [43]. Since we have previously demonstrated that the spleen is one of the major tissues for HIV-1 replication in our hHSC-transplanted humanized mouse model [25], we assumed that the endogenous expression levels of APOBEC3 genes in the splenic human mononuclear cells (MNCs) affect the kinetics of 4A HIV-1 growth. As shown in Figure 3B, the expression levels of APOBEC3F and APOBEC3G were 6.4-fold and 24.7-fold higher than those of APOBEC3D, respectively, and the expression level of APOBEC3G was 3.9-fold higher than that of APOBEC3F.


APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

Sato K, Takeuchi JS, Misawa N, Izumi T, Kobayashi T, Kimura Y, Iwami S, Takaori-Kondo A, Hu WS, Aihara K, Ito M, An DS, Pathak VK, Koyanagi Y - PLoS Pathog. (2014)

Expression levels of APOBEC3 and IFNB in infected humanized mice.(A) Dispersion of HIV-1 growth efficiency in humanized mice. (Left) The peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) were classified into 4 degrees (less than 103, 103–104, 104–105, or more than 105), and the distribution is plotted. (Right) The coefficient of variance of the peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) is shown. (B) Expression levels of APOBEC3D, APOBEC3F, and APOBEC3G in the splenic human CD4+ T cells of humanized mice (n = 73) were analyzed by real-time RT-PCR. The values were standardized as previously described [43], and the level of APOBEC3D is set to 1 to facilitate comparison. (C) Expression levels of APOBEC3D, APOBEC3F, APOBEC3G, and IFNB in the splenic human CD4+ T cells of infected mice (WT, n = 13; 4A, n = 20; 5A, n = 17; and 4A5A, n = 15) and mock-infected mice (n = 8) at 6 wpi were analyzed by real-time RT-PCR. Horizontal bars represent the averages. Asterisks represent statistically significant difference (P<0.05 by Student's t test) between infected mice and mock-infected mice. (D) Negative correlation between VL and APOBEC3 expression in 4A HIV-1-infected humanized mice. The mRNA expression levels of APOBEC3D (left), APOBEC3F (middle), and APOBEC3G (right) in the splenic human CD4+ T cells (x-axes) and the VL at 6 wpi (y-axis) of 4A HIV-1-infected mice (n = 20) are shown. The lines represent exponential approximation. Pearson correlation coefficient (r) was adopted to determine statistically significant correlation between each value.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4199767&req=5

ppat-1004453-g003: Expression levels of APOBEC3 and IFNB in infected humanized mice.(A) Dispersion of HIV-1 growth efficiency in humanized mice. (Left) The peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) were classified into 4 degrees (less than 103, 103–104, 104–105, or more than 105), and the distribution is plotted. (Right) The coefficient of variance of the peak VLs of WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), and 4A5A HIV-1 (n = 8) is shown. (B) Expression levels of APOBEC3D, APOBEC3F, and APOBEC3G in the splenic human CD4+ T cells of humanized mice (n = 73) were analyzed by real-time RT-PCR. The values were standardized as previously described [43], and the level of APOBEC3D is set to 1 to facilitate comparison. (C) Expression levels of APOBEC3D, APOBEC3F, APOBEC3G, and IFNB in the splenic human CD4+ T cells of infected mice (WT, n = 13; 4A, n = 20; 5A, n = 17; and 4A5A, n = 15) and mock-infected mice (n = 8) at 6 wpi were analyzed by real-time RT-PCR. Horizontal bars represent the averages. Asterisks represent statistically significant difference (P<0.05 by Student's t test) between infected mice and mock-infected mice. (D) Negative correlation between VL and APOBEC3 expression in 4A HIV-1-infected humanized mice. The mRNA expression levels of APOBEC3D (left), APOBEC3F (middle), and APOBEC3G (right) in the splenic human CD4+ T cells (x-axes) and the VL at 6 wpi (y-axis) of 4A HIV-1-infected mice (n = 20) are shown. The lines represent exponential approximation. Pearson correlation coefficient (r) was adopted to determine statistically significant correlation between each value.
Mentions: In the 3 kinds of HIV-1 vif mutants, it was noteworthy that the VL in each 4A HIV-1-infected mouse varied between individual mice, while those in 5A and 4A5A HIV-1-infected mice were uniformly low (Figure 3A, left panel). In fact, the coefficient of variance of peak VL, which indicates the extent of distribution, in 4A HIV-1-infected mice was ∼2-fold higher than that in WT, 5A, and 4A5A HIV-1-infected mice (Figure 3A, right panel). These findings raised a possibility that the level of viremia in 4A HIV-1-infected mice is correlated with endogenous APOBEC3 expression levels. To address this possibility, we determined the endogenous expression levels of APOBEC3D, APOBEC3F, and APOBEC3G in the spleen of humanized mice by real-time RT-PCR and standardized them to those of APOBEC3D according to the procedure reported previously [42], [43]. Since we have previously demonstrated that the spleen is one of the major tissues for HIV-1 replication in our hHSC-transplanted humanized mouse model [25], we assumed that the endogenous expression levels of APOBEC3 genes in the splenic human mononuclear cells (MNCs) affect the kinetics of 4A HIV-1 growth. As shown in Figure 3B, the expression levels of APOBEC3F and APOBEC3G were 6.4-fold and 24.7-fold higher than those of APOBEC3D, respectively, and the expression level of APOBEC3G was 3.9-fold higher than that of APOBEC3F.

Bottom Line: Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo.We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F.Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, Kyoto, Kyoto, Japan.

ABSTRACT
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 in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

Show MeSH
Related in: MedlinePlus