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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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Association of A3H-hapII with LAI Vif, but not with NL4-3 Vif.5 µg HA-tagged A3H-hapII was co-transfected with 1 µg of the indicated Vif expression plasmids in 293T cells in 10-cm dishes. Cells were treated with clasto-Lactacystin β-lactone (10 µM) for ten hours and cells were lysed in mild lysis buffer 2 days post transfection. Lysates were cleared and incubated with anti-HA tagged beads (Sigma) for one hour at 4°C. Beads were extensively washed with lysis buffer and proteins were eluted by boiling in sample loading buffer. Proteins were analyzed by western blot.
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pone-0057744-g004: Association of A3H-hapII with LAI Vif, but not with NL4-3 Vif.5 µg HA-tagged A3H-hapII was co-transfected with 1 µg of the indicated Vif expression plasmids in 293T cells in 10-cm dishes. Cells were treated with clasto-Lactacystin β-lactone (10 µM) for ten hours and cells were lysed in mild lysis buffer 2 days post transfection. Lysates were cleared and incubated with anti-HA tagged beads (Sigma) for one hour at 4°C. Beads were extensively washed with lysis buffer and proteins were eluted by boiling in sample loading buffer. Proteins were analyzed by western blot.

Mentions: The observation that A3H-hapII is sensitive to LAI Vif but not NL4-3 Vif could be explained by a difference in Vif binding to A3H-hapII. We studied the interaction of A3H-hapII with both Vif variants by co-immunoprecipitation. HA-tagged A3H-hapII was co-transfected with empty pCRV1 plasmids or pCRV1 expressing NL4-3 and LAI Vif in 293T cells. To block A3H degradation by LAI Vif, the proteasome inhibitor, clasto-Lactacystin β-lactone, was added to the transfected HEK 293T cells 24 hours prior to cell lysis. Cleared lysates were incubated with α-HA coated beads and extensively washed with lysis buffer and eluted. Western blot analysis of the cell lysates showed that Vifs and A3H were equally expressed and that A3H-hapII was not degraded by LAI Vif in the presence of proteasome inhibitor (Figure 4). LAI Vif co-precipitated efficiently with A3H-hapII, whereas only very little NL4-3 Vif was bound (Figure 4). Together, this indicates that LAI Vif, but not NL4-3 Vif, can efficiently associate with A3H-hapII. This ability of LAI Vif correlates with its efficiency to degrade and counteract the antiviral activity of A3H-hapII.


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)

Association of A3H-hapII with LAI Vif, but not with NL4-3 Vif.5 µg HA-tagged A3H-hapII was co-transfected with 1 µg of the indicated Vif expression plasmids in 293T cells in 10-cm dishes. Cells were treated with clasto-Lactacystin β-lactone (10 µM) for ten hours and cells were lysed in mild lysis buffer 2 days post transfection. Lysates were cleared and incubated with anti-HA tagged beads (Sigma) for one hour at 4°C. Beads were extensively washed with lysis buffer and proteins were eluted by boiling in sample loading buffer. Proteins were analyzed by western blot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585137&req=5

pone-0057744-g004: Association of A3H-hapII with LAI Vif, but not with NL4-3 Vif.5 µg HA-tagged A3H-hapII was co-transfected with 1 µg of the indicated Vif expression plasmids in 293T cells in 10-cm dishes. Cells were treated with clasto-Lactacystin β-lactone (10 µM) for ten hours and cells were lysed in mild lysis buffer 2 days post transfection. Lysates were cleared and incubated with anti-HA tagged beads (Sigma) for one hour at 4°C. Beads were extensively washed with lysis buffer and proteins were eluted by boiling in sample loading buffer. Proteins were analyzed by western blot.
Mentions: The observation that A3H-hapII is sensitive to LAI Vif but not NL4-3 Vif could be explained by a difference in Vif binding to A3H-hapII. We studied the interaction of A3H-hapII with both Vif variants by co-immunoprecipitation. HA-tagged A3H-hapII was co-transfected with empty pCRV1 plasmids or pCRV1 expressing NL4-3 and LAI Vif in 293T cells. To block A3H degradation by LAI Vif, the proteasome inhibitor, clasto-Lactacystin β-lactone, was added to the transfected HEK 293T cells 24 hours prior to cell lysis. Cleared lysates were incubated with α-HA coated beads and extensively washed with lysis buffer and eluted. Western blot analysis of the cell lysates showed that Vifs and A3H were equally expressed and that A3H-hapII was not degraded by LAI Vif in the presence of proteasome inhibitor (Figure 4). LAI Vif co-precipitated efficiently with A3H-hapII, whereas only very little NL4-3 Vif was bound (Figure 4). Together, this indicates that LAI Vif, but not NL4-3 Vif, can efficiently associate with A3H-hapII. This ability of LAI Vif correlates with its efficiency to degrade and counteract the antiviral activity of A3H-hapII.

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