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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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Vif 48H is required for A3H hapII degradation and rescue of infectivity.(A) Schematic overview of the first 61 amino acids of NL4-3 and LAI Vif. Three regions that are different between NL4-3 and LAI are indicated by black boxes. (B) 50 ng NL4-3 or LAI or mutant Vif expression plasmids was co-transfected with 100 ng of FLAG-tagged A3H-hapII in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (C) A3H-hapII restriction of Vif proficient mutant Vif and Vif deficient HIV-1 NL4-3 and LAI. 50 ng A3H-hapII was co-transfected with the different indicated HIV molecular clones (500 ng) in 293T cells. Two days post infection cleared supernatants were used to infect TZM-bl reporter cells and β-Galactosidase was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections.
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pone-0057744-g005: Vif 48H is required for A3H hapII degradation and rescue of infectivity.(A) Schematic overview of the first 61 amino acids of NL4-3 and LAI Vif. Three regions that are different between NL4-3 and LAI are indicated by black boxes. (B) 50 ng NL4-3 or LAI or mutant Vif expression plasmids was co-transfected with 100 ng of FLAG-tagged A3H-hapII in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (C) A3H-hapII restriction of Vif proficient mutant Vif and Vif deficient HIV-1 NL4-3 and LAI. 50 ng A3H-hapII was co-transfected with the different indicated HIV molecular clones (500 ng) in 293T cells. Two days post infection cleared supernatants were used to infect TZM-bl reporter cells and β-Galactosidase was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections.

Mentions: NL4-3 and LAI Vifs only differ at nine positions in the N-terminal region (see Figure 5A), which is the portion of Vif critical for interaction with several APOBEC3 proteins. In order to pinpoint the differential anti-A3H activity at a single residue level, we generated a set of Vif chimeras in which LAI Vif amino acids were introduced into NL4-3 Vif (Figure 5A). The A3H-hapII degradation efficiency in the presence of the WT NL4-3, WT LAI and the chimeras was analyzed by western blot. The introduction of either LAI amino acids RS or VGRG into NL4-3 Vif did not results in an increase in activity, indicating that these regions are dispensable for specific A3H-hapII recognition (Figure 5B). However, a NL4-3 Vif variant with amino acids PHR efficiently degraded A3H-hapII. Additional single amino acid changes within the PHR stretch revealed that the introduction of a histidine at position 48 was sufficient to fully confer activity towards A3H-hapII. Indeed, the reverse change in LAI Vif, H48N, resulted in a complete loss of A3H-hapII degradation activity (Figure 5B). We next replaced the Vif position 48 in the full-length molecular clones NL4-3 and LAI and tested their infectivity levels in the presence of A3H-hapII (Figure 5C). Both NL4-3 WT and NL4-3 ΔVif were restricted, but the restriction was clearly relieved for NL4-3 N48H. Conversely, LAI WT displayed high infectivity levels, but the H48N change efficiently restricted LAI H48N, similar to LAI ΔVif. In summary, the difference in counteracting A3H-hapII between NL4-3 and LAI Vif can be attributed to a single amino acid at position 48.


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)

Vif 48H is required for A3H hapII degradation and rescue of infectivity.(A) Schematic overview of the first 61 amino acids of NL4-3 and LAI Vif. Three regions that are different between NL4-3 and LAI are indicated by black boxes. (B) 50 ng NL4-3 or LAI or mutant Vif expression plasmids was co-transfected with 100 ng of FLAG-tagged A3H-hapII in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (C) A3H-hapII restriction of Vif proficient mutant Vif and Vif deficient HIV-1 NL4-3 and LAI. 50 ng A3H-hapII was co-transfected with the different indicated HIV molecular clones (500 ng) in 293T cells. Two days post infection cleared supernatants were used to infect TZM-bl reporter cells and β-Galactosidase was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections.
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pone-0057744-g005: Vif 48H is required for A3H hapII degradation and rescue of infectivity.(A) Schematic overview of the first 61 amino acids of NL4-3 and LAI Vif. Three regions that are different between NL4-3 and LAI are indicated by black boxes. (B) 50 ng NL4-3 or LAI or mutant Vif expression plasmids was co-transfected with 100 ng of FLAG-tagged A3H-hapII in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (C) A3H-hapII restriction of Vif proficient mutant Vif and Vif deficient HIV-1 NL4-3 and LAI. 50 ng A3H-hapII was co-transfected with the different indicated HIV molecular clones (500 ng) in 293T cells. Two days post infection cleared supernatants were used to infect TZM-bl reporter cells and β-Galactosidase was measured two days later. One representative experiment consisting of triplicate transfections is shown. Error bars represent standard deviations from triplicate transfections.
Mentions: NL4-3 and LAI Vifs only differ at nine positions in the N-terminal region (see Figure 5A), which is the portion of Vif critical for interaction with several APOBEC3 proteins. In order to pinpoint the differential anti-A3H activity at a single residue level, we generated a set of Vif chimeras in which LAI Vif amino acids were introduced into NL4-3 Vif (Figure 5A). The A3H-hapII degradation efficiency in the presence of the WT NL4-3, WT LAI and the chimeras was analyzed by western blot. The introduction of either LAI amino acids RS or VGRG into NL4-3 Vif did not results in an increase in activity, indicating that these regions are dispensable for specific A3H-hapII recognition (Figure 5B). However, a NL4-3 Vif variant with amino acids PHR efficiently degraded A3H-hapII. Additional single amino acid changes within the PHR stretch revealed that the introduction of a histidine at position 48 was sufficient to fully confer activity towards A3H-hapII. Indeed, the reverse change in LAI Vif, H48N, resulted in a complete loss of A3H-hapII degradation activity (Figure 5B). We next replaced the Vif position 48 in the full-length molecular clones NL4-3 and LAI and tested their infectivity levels in the presence of A3H-hapII (Figure 5C). Both NL4-3 WT and NL4-3 ΔVif were restricted, but the restriction was clearly relieved for NL4-3 N48H. Conversely, LAI WT displayed high infectivity levels, but the H48N change efficiently restricted LAI H48N, similar to LAI ΔVif. In summary, the difference in counteracting A3H-hapII between NL4-3 and LAI Vif can be attributed to a single amino acid at position 48.

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