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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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Diversification and functional evolution of 4A HIV-1 in vivo.The env ORFs (6221–8782, 2,562 bases) of viral RNA in the plasma of infected mice (WT, n = 73 from 2 mice; 4A, n = 91 from 3 mice; 5A, n = 68 from 2 mice; and 4A5A, n = 33 from 1 mouse) were sequenced by SGS assay. Raw data are shown in Figure S7. (A) The mutation matrix (top) and the pie chart of G-to-A mutation (bottom) are shown. In the bottom panel, the diameters of pie charts represent the percentage of G-to-A mutations in total mutations. (B) Effect of G-to-A mutation in env ORF of viral RNA in plasma. Pie chart of the effect of G-to-A mutation in env ORF is shown. The numbers in pie chart represent the percentage of termination, nonsynonymous, and synonymous mutations in G-to-A mutations, respectively. (C and D) Divergence of viral RNA sequence. Phylogenic trees (C) and genetic diversity (D) of env ORF sequences in the plasma of infected mice are shown. In panel C, the scale bar indicates the number of substitutions per site. The amplicons harboring statistically significant levels of G-to-A mutations (P<0.05 by Fisher's exact test using Hypermut 2.0) are indicated by asterisks.
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ppat-1004453-g005: Diversification and functional evolution of 4A HIV-1 in vivo.The env ORFs (6221–8782, 2,562 bases) of viral RNA in the plasma of infected mice (WT, n = 73 from 2 mice; 4A, n = 91 from 3 mice; 5A, n = 68 from 2 mice; and 4A5A, n = 33 from 1 mouse) were sequenced by SGS assay. Raw data are shown in Figure S7. (A) The mutation matrix (top) and the pie chart of G-to-A mutation (bottom) are shown. In the bottom panel, the diameters of pie charts represent the percentage of G-to-A mutations in total mutations. (B) Effect of G-to-A mutation in env ORF of viral RNA in plasma. Pie chart of the effect of G-to-A mutation in env ORF is shown. The numbers in pie chart represent the percentage of termination, nonsynonymous, and synonymous mutations in G-to-A mutations, respectively. (C and D) Divergence of viral RNA sequence. Phylogenic trees (C) and genetic diversity (D) of env ORF sequences in the plasma of infected mice are shown. In panel C, the scale bar indicates the number of substitutions per site. The amplicons harboring statistically significant levels of G-to-A mutations (P<0.05 by Fisher's exact test using Hypermut 2.0) are indicated by asterisks.

Mentions: Our findings in both in vivo (Figure 4F) and in vitro (Figure 4G) demonstrated that APOBEC3G prefers to target TGGG as substrate. Importantly, TGG and TAG are the codons encoding Tryptophan and termination codon, respectively, suggesting that APOBEC3G can readily cause lethal mutations (i.e., TGG-to-TAG termination mutations). On the other hand, APOBEC3F and APOBEC3D generated GAA-to-AAA and GA-to-AA mutations, respectively (Figures 4F and 4G), which do not generate termination codons and thus cause lethal mutations less frequently. These findings raised a hypothesis that APOBEC3G directly causes lethal mutations, while APOBEC3F and APOBEC3D induce the accumulation of nonsynonymous mutations in the viral genome. To address this hypothesis, single genome sequencing (SGS) assays [50] were performed using viral RNA isolated from the plasma of infected mice at 6 wpi. Since G-to-A mutations were frequently observed in the proximal upstream region of the 3′ polypurine tract (positioned at 9056–9071; Figure S6), which was consistent with previous reports [49], [51], we focused on the env open reading frame (ORF) sequence. As shown in Figure 5A (the raw data is shown in Figure S7), SGS assay revealed that G-to-A mutations were frequently observed in the viral RNA genomes of 4A HIV-1-infected mice but not of WT, 5A, and 4A5A HIV-1-infected mice. In addition, in the 91 env amplicons of 4A HIV-1-infected mice, 37 analyzed amplicons harbored more than 10 G-to-A mutations, and 15 analyzed amplicons harbored more than 10 GA-to-AA mutations, respectively (Figure S8). On the other hand, the amplicons harboring G-to-A hypermutations were rarely detected in WT, 5A, and 4A5A HIV-1-infected mice (Figure S8). Moreover, although termination mutations were prominently detected in the proviral DNA of 5A and 4A5A HIV-1-infected mice (Figures 4D and 4E), the percentages of termination mutation in the viral RNA in plasma of 5A and 4A5A HIV-1-infected mice were comparable to that of 4A HIV-1-infected mice (Figure 5B; 4A HIV-1 versus 5A HIV-1, P = 0.06; 4A HIV-1 versus 4A5A HIV-1, P = 0.19 by Chi-square test for independence). These findings strongly suggest that APOBEC3G-mediated G-to-A mutations frequently result in lethal mutations.


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)

Diversification and functional evolution of 4A HIV-1 in vivo.The env ORFs (6221–8782, 2,562 bases) of viral RNA in the plasma of infected mice (WT, n = 73 from 2 mice; 4A, n = 91 from 3 mice; 5A, n = 68 from 2 mice; and 4A5A, n = 33 from 1 mouse) were sequenced by SGS assay. Raw data are shown in Figure S7. (A) The mutation matrix (top) and the pie chart of G-to-A mutation (bottom) are shown. In the bottom panel, the diameters of pie charts represent the percentage of G-to-A mutations in total mutations. (B) Effect of G-to-A mutation in env ORF of viral RNA in plasma. Pie chart of the effect of G-to-A mutation in env ORF is shown. The numbers in pie chart represent the percentage of termination, nonsynonymous, and synonymous mutations in G-to-A mutations, respectively. (C and D) Divergence of viral RNA sequence. Phylogenic trees (C) and genetic diversity (D) of env ORF sequences in the plasma of infected mice are shown. In panel C, the scale bar indicates the number of substitutions per site. The amplicons harboring statistically significant levels of G-to-A mutations (P<0.05 by Fisher's exact test using Hypermut 2.0) are indicated by asterisks.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199767&req=5

ppat-1004453-g005: Diversification and functional evolution of 4A HIV-1 in vivo.The env ORFs (6221–8782, 2,562 bases) of viral RNA in the plasma of infected mice (WT, n = 73 from 2 mice; 4A, n = 91 from 3 mice; 5A, n = 68 from 2 mice; and 4A5A, n = 33 from 1 mouse) were sequenced by SGS assay. Raw data are shown in Figure S7. (A) The mutation matrix (top) and the pie chart of G-to-A mutation (bottom) are shown. In the bottom panel, the diameters of pie charts represent the percentage of G-to-A mutations in total mutations. (B) Effect of G-to-A mutation in env ORF of viral RNA in plasma. Pie chart of the effect of G-to-A mutation in env ORF is shown. The numbers in pie chart represent the percentage of termination, nonsynonymous, and synonymous mutations in G-to-A mutations, respectively. (C and D) Divergence of viral RNA sequence. Phylogenic trees (C) and genetic diversity (D) of env ORF sequences in the plasma of infected mice are shown. In panel C, the scale bar indicates the number of substitutions per site. The amplicons harboring statistically significant levels of G-to-A mutations (P<0.05 by Fisher's exact test using Hypermut 2.0) are indicated by asterisks.
Mentions: Our findings in both in vivo (Figure 4F) and in vitro (Figure 4G) demonstrated that APOBEC3G prefers to target TGGG as substrate. Importantly, TGG and TAG are the codons encoding Tryptophan and termination codon, respectively, suggesting that APOBEC3G can readily cause lethal mutations (i.e., TGG-to-TAG termination mutations). On the other hand, APOBEC3F and APOBEC3D generated GAA-to-AAA and GA-to-AA mutations, respectively (Figures 4F and 4G), which do not generate termination codons and thus cause lethal mutations less frequently. These findings raised a hypothesis that APOBEC3G directly causes lethal mutations, while APOBEC3F and APOBEC3D induce the accumulation of nonsynonymous mutations in the viral genome. To address this hypothesis, single genome sequencing (SGS) assays [50] were performed using viral RNA isolated from the plasma of infected mice at 6 wpi. Since G-to-A mutations were frequently observed in the proximal upstream region of the 3′ polypurine tract (positioned at 9056–9071; Figure S6), which was consistent with previous reports [49], [51], we focused on the env open reading frame (ORF) sequence. As shown in Figure 5A (the raw data is shown in Figure S7), SGS assay revealed that G-to-A mutations were frequently observed in the viral RNA genomes of 4A HIV-1-infected mice but not of WT, 5A, and 4A5A HIV-1-infected mice. In addition, in the 91 env amplicons of 4A HIV-1-infected mice, 37 analyzed amplicons harbored more than 10 G-to-A mutations, and 15 analyzed amplicons harbored more than 10 GA-to-AA mutations, respectively (Figure S8). On the other hand, the amplicons harboring G-to-A hypermutations were rarely detected in WT, 5A, and 4A5A HIV-1-infected mice (Figure S8). Moreover, although termination mutations were prominently detected in the proviral DNA of 5A and 4A5A HIV-1-infected mice (Figures 4D and 4E), the percentages of termination mutation in the viral RNA in plasma of 5A and 4A5A HIV-1-infected mice were comparable to that of 4A HIV-1-infected mice (Figure 5B; 4A HIV-1 versus 5A HIV-1, P = 0.06; 4A HIV-1 versus 4A5A HIV-1, P = 0.19 by Chi-square test for independence). These findings strongly suggest that APOBEC3G-mediated G-to-A mutations frequently result in lethal mutations.

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