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Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity.

Refsland EW, Hultquist JF, Luengas EM, Ikeda T, Shaban NM, Law EK, Brown WL, Reilly C, Emerman M, Harris RS - PLoS Genet. (2014)

Bottom Line: We also found that APOBEC3H protein levels are induced over 10-fold by infection.Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue.These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

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Generation and validation of HIV-1 Vif separation-of-function molecular/viral probes.A) A schematic of the Vif protein encoded by each HIV-1 molecular clone showing amino acid differences responsible for the hyper- and hypo-Vif functionality relative to lab-Vif (HIV-1 IIIB/NL4-3) against stable A3H haplotype II. B) Immunoblots showing the expression levels of the indicated A3 proteins stably expressed in SupT11 cells. In this experiment untagged A3H is detected with the mouse monoclonal antibody P3A3-A10. C) HIV-1 spreading infection kinetics for the indicated viruses on A3-expressing SupT11 cells lines described in panel B. The hyper-, lab-, and hypo-Vif isolates spread with similar kinetics on cells expressing a control vector, A3D, A3F, or A3G, but showed clear phenotypic differences on cells expressing low, intermediate (int), and high levels of stable A3H haplotype II. Delta-Vif virus replication was evident in control vector expressing SupT11 cells, delayed in A3D expressing cells, and suppressed under all other conditions (some symbols eclipsed).
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pgen-1004761-g003: Generation and validation of HIV-1 Vif separation-of-function molecular/viral probes.A) A schematic of the Vif protein encoded by each HIV-1 molecular clone showing amino acid differences responsible for the hyper- and hypo-Vif functionality relative to lab-Vif (HIV-1 IIIB/NL4-3) against stable A3H haplotype II. B) Immunoblots showing the expression levels of the indicated A3 proteins stably expressed in SupT11 cells. In this experiment untagged A3H is detected with the mouse monoclonal antibody P3A3-A10. C) HIV-1 spreading infection kinetics for the indicated viruses on A3-expressing SupT11 cells lines described in panel B. The hyper-, lab-, and hypo-Vif isolates spread with similar kinetics on cells expressing a control vector, A3D, A3F, or A3G, but showed clear phenotypic differences on cells expressing low, intermediate (int), and high levels of stable A3H haplotype II. Delta-Vif virus replication was evident in control vector expressing SupT11 cells, delayed in A3D expressing cells, and suppressed under all other conditions (some symbols eclipsed).

Mentions: In comparison to the starting virus, HIV-1 IIIB N48H, which only showed fast replication kinetics in SupT11 control vector and A3H-low expressing cells, one of the adapted viruses gained the capacity to replicate quickly in SupT11 cells expressing high levels of stable A3H. The majority (5/9) of vif sequences from this adapted population encoded a K63E amino acid substitution. Spreading infection experiments with an isogenic set of molecular clones showed that K63E combined with N48H to improve virus replication even in the presence of high levels of stable A3H (Figure 2C). Furthermore, we noticed that the K63E substitution recovered in these adaptation experiments is part of a cluster of 4 amino acids that distinguishes the IIIB lab strain from an isolate recovered from a homozygous stable A3H haplotype II patient [34]. The Vif protein from this particular isolate is even better than LAI Vif at counteracting stable A3H haplotype II [34]. We therefore predicted that the combination of N48H (to be more LAI Vif-like [42]) and GDAK60-63 to EKGE60-63 (to be more haplotype II patient Vif-like [34]) would result in a hyper-functional Vif protein capable of fully antagonizing stable A3H haplotype II (hyper-Vif; Figure 3A). In addition, the separation-of-function approach required the generation of a Vif variant even more sensitive than lab-Vif to restriction by stable A3H haplotype II. This was done by introducing F39V into the HIV-1 IIIB lab strain, which is a substitution that confers sensitivity to stable A3H haplotype II [41] (Figure 3A).


Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity.

Refsland EW, Hultquist JF, Luengas EM, Ikeda T, Shaban NM, Law EK, Brown WL, Reilly C, Emerman M, Harris RS - PLoS Genet. (2014)

Generation and validation of HIV-1 Vif separation-of-function molecular/viral probes.A) A schematic of the Vif protein encoded by each HIV-1 molecular clone showing amino acid differences responsible for the hyper- and hypo-Vif functionality relative to lab-Vif (HIV-1 IIIB/NL4-3) against stable A3H haplotype II. B) Immunoblots showing the expression levels of the indicated A3 proteins stably expressed in SupT11 cells. In this experiment untagged A3H is detected with the mouse monoclonal antibody P3A3-A10. C) HIV-1 spreading infection kinetics for the indicated viruses on A3-expressing SupT11 cells lines described in panel B. The hyper-, lab-, and hypo-Vif isolates spread with similar kinetics on cells expressing a control vector, A3D, A3F, or A3G, but showed clear phenotypic differences on cells expressing low, intermediate (int), and high levels of stable A3H haplotype II. Delta-Vif virus replication was evident in control vector expressing SupT11 cells, delayed in A3D expressing cells, and suppressed under all other conditions (some symbols eclipsed).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4238949&req=5

pgen-1004761-g003: Generation and validation of HIV-1 Vif separation-of-function molecular/viral probes.A) A schematic of the Vif protein encoded by each HIV-1 molecular clone showing amino acid differences responsible for the hyper- and hypo-Vif functionality relative to lab-Vif (HIV-1 IIIB/NL4-3) against stable A3H haplotype II. B) Immunoblots showing the expression levels of the indicated A3 proteins stably expressed in SupT11 cells. In this experiment untagged A3H is detected with the mouse monoclonal antibody P3A3-A10. C) HIV-1 spreading infection kinetics for the indicated viruses on A3-expressing SupT11 cells lines described in panel B. The hyper-, lab-, and hypo-Vif isolates spread with similar kinetics on cells expressing a control vector, A3D, A3F, or A3G, but showed clear phenotypic differences on cells expressing low, intermediate (int), and high levels of stable A3H haplotype II. Delta-Vif virus replication was evident in control vector expressing SupT11 cells, delayed in A3D expressing cells, and suppressed under all other conditions (some symbols eclipsed).
Mentions: In comparison to the starting virus, HIV-1 IIIB N48H, which only showed fast replication kinetics in SupT11 control vector and A3H-low expressing cells, one of the adapted viruses gained the capacity to replicate quickly in SupT11 cells expressing high levels of stable A3H. The majority (5/9) of vif sequences from this adapted population encoded a K63E amino acid substitution. Spreading infection experiments with an isogenic set of molecular clones showed that K63E combined with N48H to improve virus replication even in the presence of high levels of stable A3H (Figure 2C). Furthermore, we noticed that the K63E substitution recovered in these adaptation experiments is part of a cluster of 4 amino acids that distinguishes the IIIB lab strain from an isolate recovered from a homozygous stable A3H haplotype II patient [34]. The Vif protein from this particular isolate is even better than LAI Vif at counteracting stable A3H haplotype II [34]. We therefore predicted that the combination of N48H (to be more LAI Vif-like [42]) and GDAK60-63 to EKGE60-63 (to be more haplotype II patient Vif-like [34]) would result in a hyper-functional Vif protein capable of fully antagonizing stable A3H haplotype II (hyper-Vif; Figure 3A). In addition, the separation-of-function approach required the generation of a Vif variant even more sensitive than lab-Vif to restriction by stable A3H haplotype II. This was done by introducing F39V into the HIV-1 IIIB lab strain, which is a substitution that confers sensitivity to stable A3H haplotype II [41] (Figure 3A).

Bottom Line: We also found that APOBEC3H protein levels are induced over 10-fold by infection.Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue.These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

Show MeSH
Related in: MedlinePlus