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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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NL4-3 Vif 48H replicates more efficiently on SupT1 cells expressing A3H-hapII.NL4-3 WT (closed circles) and NL4-3 Vif N48H (open circles) were used to infect SupT1 T-cell lines expressing untagged APOBEC3H hapII (A3H hapII, A), HA-tagged APOBEC3G (A3G, B), and the empty vector (C). Clarified supernatants were harvested each day and infectivity was determined on TZM-bl reporter cells. Representative infections of each cell lines are shown. Error bars represent standard deviations of triplicate TZM-bl infections.
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pone-0057744-g007: NL4-3 Vif 48H replicates more efficiently on SupT1 cells expressing A3H-hapII.NL4-3 WT (closed circles) and NL4-3 Vif N48H (open circles) were used to infect SupT1 T-cell lines expressing untagged APOBEC3H hapII (A3H hapII, A), HA-tagged APOBEC3G (A3G, B), and the empty vector (C). Clarified supernatants were harvested each day and infectivity was determined on TZM-bl reporter cells. Representative infections of each cell lines are shown. Error bars represent standard deviations of triplicate TZM-bl infections.

Mentions: Vif position 48 is specific for A3H recognition in single cycle infectivity assays in which APOBEC3 is expressed to high levels in the HEK 293T producer cells. To asses the impact of Vif position 48 on NL4-3 replication in a more physiological experimental system, we infected previously described SupT1 T-cell lines that express low levels of A3G or A3H hapII [16] with NL4-3 WT and NL4-3 Vif N48H. NL4-3 WT replication was delayed by an order of magnitude on SupT1 cells expressing A3H hapII (Figure 7A) indicating that NL4-3 WT fails to efficiently counteract A3H when expressed at near physiological levels in T cells [16]. However, the N48H mutation in Vif improved viral replication to levels observed with the empty vector and A3G expressing cells, indicating that N48H also efficiently counteracts A3H in the setting of spreading infections. Of note, both viruses replicated with similar efficiencies on cells expressing the control empty vector (Figure 7B). Interestingly, the replication of NL4-3 WT was slightly improved compared to the N48H mutant on A3G expressing SupT1 cells, which may indicate that a Vif encoding 48N is somewhat better adapted to counteract A3G (Figure 7B). Taken together, the data indicate that Vif position 48 is specific for counteracting A3H in both single cycle as well as multiple round infection models.


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)

NL4-3 Vif 48H replicates more efficiently on SupT1 cells expressing A3H-hapII.NL4-3 WT (closed circles) and NL4-3 Vif N48H (open circles) were used to infect SupT1 T-cell lines expressing untagged APOBEC3H hapII (A3H hapII, A), HA-tagged APOBEC3G (A3G, B), and the empty vector (C). Clarified supernatants were harvested each day and infectivity was determined on TZM-bl reporter cells. Representative infections of each cell lines are shown. Error bars represent standard deviations of triplicate TZM-bl infections.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585137&req=5

pone-0057744-g007: NL4-3 Vif 48H replicates more efficiently on SupT1 cells expressing A3H-hapII.NL4-3 WT (closed circles) and NL4-3 Vif N48H (open circles) were used to infect SupT1 T-cell lines expressing untagged APOBEC3H hapII (A3H hapII, A), HA-tagged APOBEC3G (A3G, B), and the empty vector (C). Clarified supernatants were harvested each day and infectivity was determined on TZM-bl reporter cells. Representative infections of each cell lines are shown. Error bars represent standard deviations of triplicate TZM-bl infections.
Mentions: Vif position 48 is specific for A3H recognition in single cycle infectivity assays in which APOBEC3 is expressed to high levels in the HEK 293T producer cells. To asses the impact of Vif position 48 on NL4-3 replication in a more physiological experimental system, we infected previously described SupT1 T-cell lines that express low levels of A3G or A3H hapII [16] with NL4-3 WT and NL4-3 Vif N48H. NL4-3 WT replication was delayed by an order of magnitude on SupT1 cells expressing A3H hapII (Figure 7A) indicating that NL4-3 WT fails to efficiently counteract A3H when expressed at near physiological levels in T cells [16]. However, the N48H mutation in Vif improved viral replication to levels observed with the empty vector and A3G expressing cells, indicating that N48H also efficiently counteracts A3H in the setting of spreading infections. Of note, both viruses replicated with similar efficiencies on cells expressing the control empty vector (Figure 7B). Interestingly, the replication of NL4-3 WT was slightly improved compared to the N48H mutant on A3G expressing SupT1 cells, which may indicate that a Vif encoding 48N is somewhat better adapted to counteract A3G (Figure 7B). Taken together, the data indicate that Vif position 48 is specific for counteracting A3H in both single cycle as well as multiple round infection models.

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