Catalytic activity of APOBEC3F is required for efficient restriction of Vif-deficient human immunodeficiency virus

Albin, John S.; Brown, William L.; Harris, Reuben S.

doi:10.1016/j.virol.2013.11.041

Cited by 23 publications

(19 citation statements)

References 48 publications

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“…Since H9 cells express similar levels of A3F and A3DE than A3.01 cells (Dang et al, 2006; Rose et al, 2005), we infer that they also do not significantly contribute to the non-permissive phenotype in H9 cells. This conclusion is consistent with our previous observation that stably expressed A3F has overall negligible antiviral activity when compared to A3G (Miyagi et al, 2010) although there is no unified opinion on that in the literature (Albin et al, 2014; Bishop et al, 2004; Chaipan et al, 2013; Holmes et al, 2007; Liddament et al, 2004; Mulder et al, 2010). (ii) Increasing A3G expression in A3.01 cells to levels comparable to H9 or CD4+ T cells using wild type A3G effectively blocked replication of Vif-null HIV-1 suggesting that a relatively modest increase in A3G expression is sufficient to completely restrict Vif-null virus (Fig.…”

Section: Discussionsupporting

confidence: 91%

Long-term passage of Vif-null HIV-1 in CD4 + T cells expressing sub-lethal levels of APOBEC proteins fails to develop APOBEC resistance

et al. 2017

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APOBEC3G (A3G) is a cytidine deaminase with potent antiviral activity that is antagonized by Vif. A3G is expressed in a cell type-specific manner and some semi-permissive cells, including A3.01, express A3G but fail to block replication of Vif-null HIV-1. Here we explored the semi-permissive nature of A3.01 cells and found it to be defined exclusively by the levels of A3G. Indeed, minor changes in A3G levels rendered A3.01 cells either fully permissive or non-permissive for Vif-null HIV-1. Our data indicate that A3.01 cells express sub-lethal levels of catalytically active A3G that affects Vif-null HIV-1 at the proviral level but does not completely block virus replication due to purifying selection. Attempts to use the selective pressure exerted by such sub-lethal levels of A3G to select for APOBEC-resistant Vif-null virus capable of replicating in H9 cells failed despite passaging virus for five months, demonstrating that Vif is a critical viral accessory protein.

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Section: Discussionsupporting

confidence: 91%

Long-term passage of Vif-null HIV-1 in CD4 + T cells expressing sub-lethal levels of APOBEC proteins fails to develop APOBEC resistance

et al. 2017

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“…The virus replicated slowly, and viral loads at 6 weeks were reduced by 80% relative to those of the wild type, NLCSFV3. While these findings strongly suggest a significant impact on HIV-1 replication by A3F, the interpretation of the result is not straightforward because the tetra-alanine mutation may impact other Vif activities in addition to A3F degradation (58).…”

Section: Discussionmentioning

confidence: 90%

APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication

Krisko

Begum

Baker

et al. 2016

J Virol

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The multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1 JRCSF (JRCSF) expressing a vif gene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing a vif gene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF with vif mutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSF⌬vif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforced vif mutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/ 43D replicated after a prolonged delay with no mutations in vif but instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations in pol viral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA. IMPORTANCEVif degradation of A3G and A3F enhances viral fitness, as virus with even a partially restored capacity for degradation outgrows JRCSFvifH42/43D and JRCSFvifW79S. Unexpectedly, fixation of mutations that replaced H42/43D or W79S in viral RNA lagged behind the appearance of high viral loads. In one exceptional JRCSFvifH42/43D infection, vif was unchanged but replication proceeded after a long delay. These results suggest that Vif binds and inhibits the non-cytosine deaminase activities of intact A3G and intact A3F, allowing JRCSFvifH42/43D and JRCSFvifW79S to replicate with reduced fitness. Subsequently, enhanced Vif function is acquired by enforced mutations. In infected cells, JRCSF⌬vif and JRCSFvifH42/43DW79S are exposed to active A3F and A3G and fail to replicate. JRCSFvifH42/43D Vif degrades A3F and, in some cases, overcomes A3G mutagenic activity to replicate. Vif may have evolved to inhibit A3F and A3G by stoichiometric binding and subsequently acquired the ability to target these proteins to proteasomes.

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“…The biggest surprise is that a high-resolution structure was obtained without zinc in the active site. All previous data of APOBEC crystal and NMR structures [9, 10, 12–22, 34, 35], as well as structures of phylogenetically related enzymes (CD, CDD1, ADAR, ADAT [36–51]), indicate zinc coordination at the active site.. For all tested enzymes, mutagenesis studies have shown that the conserved zinc coordinating residues are essential for catalytic activity ( e.g ., A3F H249, E251, C280, and C283 [4–6]). However, the active site glutamic acid may be dispensable for zinc binding because changing this residue in A3A (E72A) or in the distantly related Blasticidin S. deaminase (E56Q) did not prevent the protein from coordinating zinc as evidenced by crystal structures [9, 52].…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis studies have demonstrated that conserved amino acids in the zinc-coordinating motif are essential for catalysis (reviews cited above). For instance, mutating the catalytic glutamate or the zinc-coordinating residues (three cysteines or, in some enzymes, two cysteines and one histidine) completely abolishes catalytic activity and, in most instances, also abrogates HIV-1 restriction activity ( e.g ., studies on A3F [4–6]).…”

Section: Introductionmentioning

confidence: 99%

1.92 Angstrom Zinc-Free APOBEC3F Catalytic Domain Crystal Structure

Shaban

Shi

et al. 2016

Journal of Molecular Biology

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The APOBEC3 family of DNA cytosine deaminases is capable of restricting the replication of HIV-1 and other pathogens. Here we report a 1.92 angstrom resolution crystal structure of the Vif-binding and catalytic domain of APOBEC3F (A3F). This structure is distinct from previously published APOBEC and phylogentically related deaminase structures, as it is the first without zinc in the active site. We determined an additional structure containing zinc in the same crystal form that allows direct comparison with the zinc-free structure. In the absence of zinc, the conserved active site residues that normally participate in zinc coordination show unique conformations, including a 90 degree rotation of His249 and disulfide bond formation between Cys280 and Cys283. We found that zinc coordination is influenced by pH and treating the protein at low pH in crystallization buffer is sufficient to remove zinc. Zinc coordination and catalytic activity is reconstituted with the addition of zinc only in a reduced environment likely due to the two active site cysteines readily forming a disulfide bond when not coordinating zinc. We show that the enzyme is active in the presence of zinc and cobalt, but not with other divalent metals. These results unexpectedly demonstrate that zinc is not required for the structural integrity of A3F and suggest that metal coordination may be a strategy for regulating the activity of A3F and related deaminases.

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Catalytic activity of APOBEC3F is required for efficient restriction of Vif-deficient human immunodeficiency virus

Cited by 23 publications

References 48 publications

Long-term passage of Vif-null HIV-1 in CD4 + T cells expressing sub-lethal levels of APOBEC proteins fails to develop APOBEC resistance

Long-term passage of Vif-null HIV-1 in CD4 + T cells expressing sub-lethal levels of APOBEC proteins fails to develop APOBEC resistance

APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication

1.92 Angstrom Zinc-Free APOBEC3F Catalytic Domain Crystal Structure

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