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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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Functional evolution of 4A HIV-1 in vivo.(A) Mutations in env ORFs. (B) Estimation of coreceptor usage. Putative coreceptor usage was determined by using a geno2pheno coreceptor algorithm [54] as described in Materials and Methods. Mouse IDs are shown in parentheses (correspond to those in Table S2). The frequency of putative CXCR4-tropic HIV-1 in 4A HIV-1-infected mice was significantly higher than those in the mice infected with the other viruses (P = 0.043 by Chi-square test for independence). (C) Functional evaluation of coreceptor usage. Viral infectivity was measured by MaRBLE assay using R5-MaRBLE cells (left) and X4-MaRBLE cells (right). The data represents average with SD. The assay was performed in triplicate. Asterisks represent statistically significant differences (P<0.05 by Student's t test). NS, no statistical significance.
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ppat-1004453-g006: Functional evolution of 4A HIV-1 in vivo.(A) Mutations in env ORFs. (B) Estimation of coreceptor usage. Putative coreceptor usage was determined by using a geno2pheno coreceptor algorithm [54] as described in Materials and Methods. Mouse IDs are shown in parentheses (correspond to those in Table S2). The frequency of putative CXCR4-tropic HIV-1 in 4A HIV-1-infected mice was significantly higher than those in the mice infected with the other viruses (P = 0.043 by Chi-square test for independence). (C) Functional evaluation of coreceptor usage. Viral infectivity was measured by MaRBLE assay using R5-MaRBLE cells (left) and X4-MaRBLE cells (right). The data represents average with SD. The assay was performed in triplicate. Asterisks represent statistically significant differences (P<0.05 by Student's t test). NS, no statistical significance.

Mentions: As shown in Figure 6A, mutations were detected in both conserved and variable regions of env. Previous studies have demonstrated that the variable region 3 (V3) of env, particularly the residues positioned at 11 and 25 in the V3, determines the CCR5 or CXCR4 coreceptor usage for HIV-1 entry [52], [53]. Since we detected the diversified env sequences particularly in 4A HIV-1-infected mice (Figure 5C), we hypothesized the emergence of viruses that can use CXCR4 as the coreceptor in 4A HIV-1-infected mice. To address this possibility, we screened putative CXCR4-tropic HIV-1 by using a geno2pheno tool, which predicts the coreceptor usage based on nucleotide sequence [54], and found that the frequency of putative CXCR4-tropic HIV-1 in 4A HIV-1-infected mice was significantly higher than those in mice infected with WT, 5A, and 4A5A HIV-1s (Figure 6B, left). The detected putative CXCR4-tropic viruses were a N7S mutant from a WT HIV-1-infected mouse, a G24R mutant from a 4A HIV-1-infected mouse, and five E25K mutants from three 4A HIV-1-infected and one 4A5A HIV-1-infected mice (Figure 6B, right). It was particularly noteworthy that the E25K mutant detected was due to GAA-to-AAA mutation, which is the mutation signature mediated by APOBEC3F and APOBEC3D. To functionally evaluate whether these mutants can use CXCR4 as the coreceptor, we prepared the mutated virus based on NLCSFV3, which exclusively use CCR5 as the coreceptor. As shown in Figure 6C, we directly demonstrated that the infectivity of E25K mutant in CXCR4+ MaRBLE cells was 2.5-fold higher than that of parental NLCSFV3 with a statistical significance (P = 0.0073). Taken together, these findings strongly suggest that the G-to-A mutation mediated by APOBEC3F and APOBEC3D can contribute to the conversion of viral coreceptor usage from CCR5 to CXCR4.


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)

Functional evolution of 4A HIV-1 in vivo.(A) Mutations in env ORFs. (B) Estimation of coreceptor usage. Putative coreceptor usage was determined by using a geno2pheno coreceptor algorithm [54] as described in Materials and Methods. Mouse IDs are shown in parentheses (correspond to those in Table S2). The frequency of putative CXCR4-tropic HIV-1 in 4A HIV-1-infected mice was significantly higher than those in the mice infected with the other viruses (P = 0.043 by Chi-square test for independence). (C) Functional evaluation of coreceptor usage. Viral infectivity was measured by MaRBLE assay using R5-MaRBLE cells (left) and X4-MaRBLE cells (right). The data represents average with SD. The assay was performed in triplicate. Asterisks represent statistically significant differences (P<0.05 by Student's t test). NS, no statistical significance.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199767&req=5

ppat-1004453-g006: Functional evolution of 4A HIV-1 in vivo.(A) Mutations in env ORFs. (B) Estimation of coreceptor usage. Putative coreceptor usage was determined by using a geno2pheno coreceptor algorithm [54] as described in Materials and Methods. Mouse IDs are shown in parentheses (correspond to those in Table S2). The frequency of putative CXCR4-tropic HIV-1 in 4A HIV-1-infected mice was significantly higher than those in the mice infected with the other viruses (P = 0.043 by Chi-square test for independence). (C) Functional evaluation of coreceptor usage. Viral infectivity was measured by MaRBLE assay using R5-MaRBLE cells (left) and X4-MaRBLE cells (right). The data represents average with SD. The assay was performed in triplicate. Asterisks represent statistically significant differences (P<0.05 by Student's t test). NS, no statistical significance.
Mentions: As shown in Figure 6A, mutations were detected in both conserved and variable regions of env. Previous studies have demonstrated that the variable region 3 (V3) of env, particularly the residues positioned at 11 and 25 in the V3, determines the CCR5 or CXCR4 coreceptor usage for HIV-1 entry [52], [53]. Since we detected the diversified env sequences particularly in 4A HIV-1-infected mice (Figure 5C), we hypothesized the emergence of viruses that can use CXCR4 as the coreceptor in 4A HIV-1-infected mice. To address this possibility, we screened putative CXCR4-tropic HIV-1 by using a geno2pheno tool, which predicts the coreceptor usage based on nucleotide sequence [54], and found that the frequency of putative CXCR4-tropic HIV-1 in 4A HIV-1-infected mice was significantly higher than those in mice infected with WT, 5A, and 4A5A HIV-1s (Figure 6B, left). The detected putative CXCR4-tropic viruses were a N7S mutant from a WT HIV-1-infected mouse, a G24R mutant from a 4A HIV-1-infected mouse, and five E25K mutants from three 4A HIV-1-infected and one 4A5A HIV-1-infected mice (Figure 6B, right). It was particularly noteworthy that the E25K mutant detected was due to GAA-to-AAA mutation, which is the mutation signature mediated by APOBEC3F and APOBEC3D. To functionally evaluate whether these mutants can use CXCR4 as the coreceptor, we prepared the mutated virus based on NLCSFV3, which exclusively use CCR5 as the coreceptor. As shown in Figure 6C, we directly demonstrated that the infectivity of E25K mutant in CXCR4+ MaRBLE cells was 2.5-fold higher than that of parental NLCSFV3 with a statistical significance (P = 0.0073). Taken together, these findings strongly suggest that the G-to-A mutation mediated by APOBEC3F and APOBEC3D can contribute to the conversion of viral coreceptor usage from CCR5 to CXCR4.

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