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The resistance of human APOBEC3H to HIV-1 NL4-3 molecular clone is determined by a single amino acid in Vif.

Ooms M, Letko M, Binka M, Simon V - PLoS ONE (2013)

Bottom Line: Furthermore, the amino acid identity at position 48 only affects the degradation of A3H-hapII, whereas recognition of and activity against human A3D, A3F and A3G are only minimally affected.NL4-3 encoding 48H replicates better than NL4-3 WT (48N) in T cell-lines stably expressing A3H hapII, whereas there is no fitness difference in the absence of APOBEC3.These studies provide an explanation for the conflicting reports regarding A3H resistance to Vif mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT
Some human APOBEC3 cytidine deaminases have antiviral activity against HIV-1 and other retroviruses. The single deaminase domain APOBEC3H (A3H) enzyme is highly polymorphic and multiple A3H haplotypes have been identified. A3H haplotype II (A3H-hapII) possesses the strongest activity against HIV-1. There remains, however, uncertainty regarding the extent to which A3H-hapII is sensitive to HIV-1 Vif mediated degradation. We tested, therefore, the two different reference Vif proteins widely used in previous studies. We show that A3H-hapII is resistant to NL4-3 Vif while it is efficiently degraded by LAI Vif. Co-immunoprecipitation assays demonstrate that LAI Vif, but not NL4-3 Vif associates with A3H-hapII. Chimeras between NL4-3 and LAI Vif identify the amino acid responsible for the differential degradation activity: A histidine at position 48 in Vif confers activity against A3H-hapII, while an asparagine abolishes its anti-A3H activity. Furthermore, the amino acid identity at position 48 only affects the degradation of A3H-hapII, whereas recognition of and activity against human A3D, A3F and A3G are only minimally affected. NL4-3 encoding 48H replicates better than NL4-3 WT (48N) in T cell-lines stably expressing A3H hapII, whereas there is no fitness difference in the absence of APOBEC3. These studies provide an explanation for the conflicting reports regarding A3H resistance to Vif mediated degradation.

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Schematic overview of A3H-hapI, A3H-hapII and mutants and their antiviral activity.(A) The respective amino acids at positions 105, 121 and 178 are indicated. Asterisks denote natural variants [11], [14]. (B) A3H variants (20 ng) were transfected with the indicated HIV plasmids in 293T cells. Two days later, cleared supernatants were used to infect TZM-bl cells after which β-Galactosidase activity was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections. (C) 293T cells from (B) were lysed and analyzed by western blot for A3H expression (FLAG). Tubulin served as a loading control.
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pone-0057744-g003: Schematic overview of A3H-hapI, A3H-hapII and mutants and their antiviral activity.(A) The respective amino acids at positions 105, 121 and 178 are indicated. Asterisks denote natural variants [11], [14]. (B) A3H variants (20 ng) were transfected with the indicated HIV plasmids in 293T cells. Two days later, cleared supernatants were used to infect TZM-bl cells after which β-Galactosidase activity was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections. (C) 293T cells from (B) were lysed and analyzed by western blot for A3H expression (FLAG). Tubulin served as a loading control.

Mentions: In order to determine the putative contribution of A3H haplotypes to the observed Vif phenotype, we analyzed the impact of the three amino acids differing between A3H-hapI (GKE) and A3H-hapII (RDD) on the restriction of WT NL4-3 and LAI and their Vif deleted counterparts. Previous studies showed that the G105R change promotes protein stability and antiviral activity [11], [12], whereas K121D renders A3H-hapII sensitive to Vif degradation [18]. Additional A3H haplotypes encoding other combinations of these three amino acids (e.g. hapV-RDE, hapVI-GKD and hapVII-RKE) were recently described [14]. A panel of A3H mutants comprising all the possible combinations of the three residue changes were tested for antiviral activity and Vif sensitivity [Figure 3A, [12]]. We also included the artificial A3H-hapII RED variant, which was shown to be sensitive to NL4-3 Vif [20]. We analyzed the effect of these A3H variants on infectivity of NL4-3 and LAI molecular clones with and without Vif. All A3H variants encoding 105G, including A3H-hapI, failed to restrict HIV-1 (A3H-GKE, GDE, GKD, and GDD, Figure 3B). The 105R encoding variants (RKE, RDD, RKD and RDE) restricted NL4-3 WT, NL4-3 ΔVif and LAI ΔVif irrespective of the amino acid at position 121 (Figure 3B). LAI Vif could only efficiently counteract the restriction of the 121D variants (A3H-RDD and RDE), but not that of A3H-hapII encoding 121K (A3H-RKE and RKD, Figure 3B). The A3H RED variant was also sensitive to LAI Vif but, more importantly, also showed some sensitivity to NL4-3 Vif, indicating that the glutamic acid (E) at position 121 renders A3H sensitive to both LAI and NL4-3 Vif (Figure 3B). Western blot analysis of A3H expression in 293T cells showed that all four 105G carrying A3H variants (GKE, GDE, GKD and GDD) were poorly expressed and failed to restrict HIV lacking Vif (Figure 3B and 3C). A3H RKE and RKD variants were resistant to NL4-3 and LAI Vif-mediated degradation. A3H RDD and A3H RDE were both exclusively degraded by LAI Vif, but not by NL4-3 Vif. The artificial A3H RED variant was degraded by both NL4-3 and LAI Vifs. Combined, the A3H degradation results in Figure 3C are in excellent agreement with the restriction patterns depicted in Figure 3B. Taken together, these data confirm that the amino acid at position 121 determines the sensitivity of A3H to degradation by specific Vif variants: An aspartic acid (A3H 121D) mediates A3H sensitivity to LAI Vif [18], a glutamic acid (A3H 121E) renders A3H sensitive to both LAI and NL4-3 Vif and a lysine (121K) results in resistance of A3H to both Vif alleles.


The resistance of human APOBEC3H to HIV-1 NL4-3 molecular clone is determined by a single amino acid in Vif.

Ooms M, Letko M, Binka M, Simon V - PLoS ONE (2013)

Schematic overview of A3H-hapI, A3H-hapII and mutants and their antiviral activity.(A) The respective amino acids at positions 105, 121 and 178 are indicated. Asterisks denote natural variants [11], [14]. (B) A3H variants (20 ng) were transfected with the indicated HIV plasmids in 293T cells. Two days later, cleared supernatants were used to infect TZM-bl cells after which β-Galactosidase activity was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections. (C) 293T cells from (B) were lysed and analyzed by western blot for A3H expression (FLAG). Tubulin served as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585137&req=5

pone-0057744-g003: Schematic overview of A3H-hapI, A3H-hapII and mutants and their antiviral activity.(A) The respective amino acids at positions 105, 121 and 178 are indicated. Asterisks denote natural variants [11], [14]. (B) A3H variants (20 ng) were transfected with the indicated HIV plasmids in 293T cells. Two days later, cleared supernatants were used to infect TZM-bl cells after which β-Galactosidase activity was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections. (C) 293T cells from (B) were lysed and analyzed by western blot for A3H expression (FLAG). Tubulin served as a loading control.
Mentions: In order to determine the putative contribution of A3H haplotypes to the observed Vif phenotype, we analyzed the impact of the three amino acids differing between A3H-hapI (GKE) and A3H-hapII (RDD) on the restriction of WT NL4-3 and LAI and their Vif deleted counterparts. Previous studies showed that the G105R change promotes protein stability and antiviral activity [11], [12], whereas K121D renders A3H-hapII sensitive to Vif degradation [18]. Additional A3H haplotypes encoding other combinations of these three amino acids (e.g. hapV-RDE, hapVI-GKD and hapVII-RKE) were recently described [14]. A panel of A3H mutants comprising all the possible combinations of the three residue changes were tested for antiviral activity and Vif sensitivity [Figure 3A, [12]]. We also included the artificial A3H-hapII RED variant, which was shown to be sensitive to NL4-3 Vif [20]. We analyzed the effect of these A3H variants on infectivity of NL4-3 and LAI molecular clones with and without Vif. All A3H variants encoding 105G, including A3H-hapI, failed to restrict HIV-1 (A3H-GKE, GDE, GKD, and GDD, Figure 3B). The 105R encoding variants (RKE, RDD, RKD and RDE) restricted NL4-3 WT, NL4-3 ΔVif and LAI ΔVif irrespective of the amino acid at position 121 (Figure 3B). LAI Vif could only efficiently counteract the restriction of the 121D variants (A3H-RDD and RDE), but not that of A3H-hapII encoding 121K (A3H-RKE and RKD, Figure 3B). The A3H RED variant was also sensitive to LAI Vif but, more importantly, also showed some sensitivity to NL4-3 Vif, indicating that the glutamic acid (E) at position 121 renders A3H sensitive to both LAI and NL4-3 Vif (Figure 3B). Western blot analysis of A3H expression in 293T cells showed that all four 105G carrying A3H variants (GKE, GDE, GKD and GDD) were poorly expressed and failed to restrict HIV lacking Vif (Figure 3B and 3C). A3H RKE and RKD variants were resistant to NL4-3 and LAI Vif-mediated degradation. A3H RDD and A3H RDE were both exclusively degraded by LAI Vif, but not by NL4-3 Vif. The artificial A3H RED variant was degraded by both NL4-3 and LAI Vifs. Combined, the A3H degradation results in Figure 3C are in excellent agreement with the restriction patterns depicted in Figure 3B. Taken together, these data confirm that the amino acid at position 121 determines the sensitivity of A3H to degradation by specific Vif variants: An aspartic acid (A3H 121D) mediates A3H sensitivity to LAI Vif [18], a glutamic acid (A3H 121E) renders A3H sensitive to both LAI and NL4-3 Vif and a lysine (121K) results in resistance of A3H to both Vif alleles.

Bottom Line: Furthermore, the amino acid identity at position 48 only affects the degradation of A3H-hapII, whereas recognition of and activity against human A3D, A3F and A3G are only minimally affected.NL4-3 encoding 48H replicates better than NL4-3 WT (48N) in T cell-lines stably expressing A3H hapII, whereas there is no fitness difference in the absence of APOBEC3.These studies provide an explanation for the conflicting reports regarding A3H resistance to Vif mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT
Some human APOBEC3 cytidine deaminases have antiviral activity against HIV-1 and other retroviruses. The single deaminase domain APOBEC3H (A3H) enzyme is highly polymorphic and multiple A3H haplotypes have been identified. A3H haplotype II (A3H-hapII) possesses the strongest activity against HIV-1. There remains, however, uncertainty regarding the extent to which A3H-hapII is sensitive to HIV-1 Vif mediated degradation. We tested, therefore, the two different reference Vif proteins widely used in previous studies. We show that A3H-hapII is resistant to NL4-3 Vif while it is efficiently degraded by LAI Vif. Co-immunoprecipitation assays demonstrate that LAI Vif, but not NL4-3 Vif associates with A3H-hapII. Chimeras between NL4-3 and LAI Vif identify the amino acid responsible for the differential degradation activity: A histidine at position 48 in Vif confers activity against A3H-hapII, while an asparagine abolishes its anti-A3H activity. Furthermore, the amino acid identity at position 48 only affects the degradation of A3H-hapII, whereas recognition of and activity against human A3D, A3F and A3G are only minimally affected. NL4-3 encoding 48H replicates better than NL4-3 WT (48N) in T cell-lines stably expressing A3H hapII, whereas there is no fitness difference in the absence of APOBEC3. These studies provide an explanation for the conflicting reports regarding A3H resistance to Vif mediated degradation.

Show MeSH
Related in: MedlinePlus