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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 position 48 is specific for A3H-hapII.(A) 500 ng NL4-3, NL4-3 ΔVif, NL4-3 48H and (B) LAI, LAI ΔVif and LAI 48N was co-transfected with 20 ng of the different HA-tagged APOBEC3 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-g006: Vif position 48 is specific for A3H-hapII.(A) 500 ng NL4-3, NL4-3 ΔVif, NL4-3 48H and (B) LAI, LAI ΔVif and LAI 48N was co-transfected with 20 ng of the different HA-tagged APOBEC3 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: To test whether Vif position 48 affected the neutralization of other APOBEC3 proteins in addition to A3H-hapII, we analyzed the infectivity levels of WT NL4-3, and LAI as well as the corresponding Vif deleted and 48 substituted molecular clones in the presence of all seven human APOBEC3 members. NL4-3 ΔVif and LAI ΔVif were both restricted by A3B, A3D, A3F, A3G and A3H hapII to varying degrees (Figure 6). The restriction by A3D, A3F and A3G was relieved by all the Vif variants. Both LAI and NL4-3 48H Vif efficiently counteracted A3H-hapII, whereas NL4-3 and LAI 48N showed low infectivity in the presence of A3H-hapII. In these single cycle infectivity experiments, the Vif 48 mutants in both NL4-3 and LAI molecular backgrounds counteracted A3D, A3F and A3G as efficiently as the parental clones indicating that the Vif substitution at position 48 specifically affects A3H recognition without impacting other APOBEC3 proteins.


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 position 48 is specific for A3H-hapII.(A) 500 ng NL4-3, NL4-3 ΔVif, NL4-3 48H and (B) LAI, LAI ΔVif and LAI 48N was co-transfected with 20 ng of the different HA-tagged APOBEC3 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585137&req=5

pone-0057744-g006: Vif position 48 is specific for A3H-hapII.(A) 500 ng NL4-3, NL4-3 ΔVif, NL4-3 48H and (B) LAI, LAI ΔVif and LAI 48N was co-transfected with 20 ng of the different HA-tagged APOBEC3 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: To test whether Vif position 48 affected the neutralization of other APOBEC3 proteins in addition to A3H-hapII, we analyzed the infectivity levels of WT NL4-3, and LAI as well as the corresponding Vif deleted and 48 substituted molecular clones in the presence of all seven human APOBEC3 members. NL4-3 ΔVif and LAI ΔVif were both restricted by A3B, A3D, A3F, A3G and A3H hapII to varying degrees (Figure 6). The restriction by A3D, A3F and A3G was relieved by all the Vif variants. Both LAI and NL4-3 48H Vif efficiently counteracted A3H-hapII, whereas NL4-3 and LAI 48N showed low infectivity in the presence of A3H-hapII. In these single cycle infectivity experiments, the Vif 48 mutants in both NL4-3 and LAI molecular backgrounds counteracted A3D, A3F and A3G as efficiently as the parental clones indicating that the Vif substitution at position 48 specifically affects A3H recognition without impacting other APOBEC3 proteins.

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