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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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APOBEC3H sensitivity to NL4-3 and LAI Vif.(A) Increasing amounts of NL4-3 or LAI Vif expression plasmids (0, 2.5, 5, 10, 25 and 50 ng) were cotransfected with 100 ng of FLAG-tagged A3H-hapII or A3G in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (B) A3H-hapII and A3G expression from (A) were quantified by measuring non-saturated signals using the Fujifilm Intelligent Lightbox LAS-3000 instrument and Image Reader LAS-3000 software. Signals were normalized by setting both A3H-hapII and A3G expression levels without Vif at 100%. Error bars represent standard deviation from three independent experiments.
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pone-0057744-g001: APOBEC3H sensitivity to NL4-3 and LAI Vif.(A) Increasing amounts of NL4-3 or LAI Vif expression plasmids (0, 2.5, 5, 10, 25 and 50 ng) were cotransfected with 100 ng of FLAG-tagged A3H-hapII or A3G in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (B) A3H-hapII and A3G expression from (A) were quantified by measuring non-saturated signals using the Fujifilm Intelligent Lightbox LAS-3000 instrument and Image Reader LAS-3000 software. Signals were normalized by setting both A3H-hapII and A3G expression levels without Vif at 100%. Error bars represent standard deviation from three independent experiments.

Mentions: To test whether the difference in Vif sensitivity of A3H-hapII is caused by the different Vif variants, we first determined the efficiency of Vif-mediated A3H-hapII degradation in the producer cell. We co-transfected increasing amounts of NL4-3 or LAI Vif pCRV1 expression plasmids with A3H-hapII or A3G expression plasmids. A3H and A3G protein levels were analyzed two days after transfection by western blot (Figure 1A) and the intensities of the unsaturated A3H signals were quantified (Figure 1B). NL4-3 and LAI Vif showed different effects on A3H expression levels despite being expressed to similar levels (Figure 1A). NL4-3 Vif, even at high expression levels, only modestly degraded A3H-hapII, whereas small quantities of LAI Vif were sufficient for efficient A3H-hapII degradation (Figure 1B). Both Vif variants degraded A3G with similar efficiency indicating that the two Vif variants are functionally comparable with respect to A3G but specifically differ in their efficiency to degrade A3H-hapII (Figure 1A and 1B).


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)

APOBEC3H sensitivity to NL4-3 and LAI Vif.(A) Increasing amounts of NL4-3 or LAI Vif expression plasmids (0, 2.5, 5, 10, 25 and 50 ng) were cotransfected with 100 ng of FLAG-tagged A3H-hapII or A3G in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (B) A3H-hapII and A3G expression from (A) were quantified by measuring non-saturated signals using the Fujifilm Intelligent Lightbox LAS-3000 instrument and Image Reader LAS-3000 software. Signals were normalized by setting both A3H-hapII and A3G expression levels without Vif at 100%. Error bars represent standard deviation from three independent experiments.
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Related In: Results  -  Collection

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pone-0057744-g001: APOBEC3H sensitivity to NL4-3 and LAI Vif.(A) Increasing amounts of NL4-3 or LAI Vif expression plasmids (0, 2.5, 5, 10, 25 and 50 ng) were cotransfected with 100 ng of FLAG-tagged A3H-hapII or A3G in 293T cells. Two days post transfection cells were lysed and analyzed by western blot. (B) A3H-hapII and A3G expression from (A) were quantified by measuring non-saturated signals using the Fujifilm Intelligent Lightbox LAS-3000 instrument and Image Reader LAS-3000 software. Signals were normalized by setting both A3H-hapII and A3G expression levels without Vif at 100%. Error bars represent standard deviation from three independent experiments.
Mentions: To test whether the difference in Vif sensitivity of A3H-hapII is caused by the different Vif variants, we first determined the efficiency of Vif-mediated A3H-hapII degradation in the producer cell. We co-transfected increasing amounts of NL4-3 or LAI Vif pCRV1 expression plasmids with A3H-hapII or A3G expression plasmids. A3H and A3G protein levels were analyzed two days after transfection by western blot (Figure 1A) and the intensities of the unsaturated A3H signals were quantified (Figure 1B). NL4-3 and LAI Vif showed different effects on A3H expression levels despite being expressed to similar levels (Figure 1A). NL4-3 Vif, even at high expression levels, only modestly degraded A3H-hapII, whereas small quantities of LAI Vif were sufficient for efficient A3H-hapII degradation (Figure 1B). Both Vif variants degraded A3G with similar efficiency indicating that the two Vif variants are functionally comparable with respect to A3G but specifically differ in their efficiency to degrade A3H-hapII (Figure 1A and 1B).

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