Journal of Molecular Biology
Dispersed Sites of HIV Vif-Dependent Polyubiquitination in the DNA Deaminase APOBEC3F
Graphical Abstract
Highlights
► Sites of Vif-mediated polyUb in APOBEC3 proteins are not well understood. ► Residues in the N- and C-termini of A3F can act as Ub acceptor sites. ► Data suggest a flexible model for polyUb of A3 proteins by the Vif–E3 ligase complex.
Introduction
Human APOBEC3F (A3F) and APOBEC3G (A3G) are DNA cytosine deaminases capable of inhibiting human immunodeficiency virus type 1 (HIV-1) reverse transcription and integration, most prominently through their active C-terminal deaminase domains, which introduce massive levels of G-to-A mutations in the nascent provirus that contribute to incomplete reverse transcription and render hypermutated genomes hypofunctional (reviewed in Refs. [1], [2], [3]). To achieve this restriction activity, A3F and A3G must encapsidate in producer cells during viral budding, but HIV-1 typically circumvents this inhibitory APOBEC3 (A3) activity by utilizing its accessory protein Vif (virion infectivity factor) as a substrate receptor to link A3F and A3G to an E3 ubiquitin (Ub) ligase complex consisting of CBFβ, ELOB and ELOC, CUL5, and RBX2, which results in the polyubiquitination (polyUb) and subsequent proteasomal degradation of these restriction factors (see Refs. 4 and 5 and references therein).
Much effort has been devoted to identifying the determinants of the Vif–A3 interaction critical for this degradative process, including a variety of changes in Vif that result in the functional inactivation of its anti-A3 activity (e.g., Refs. 6 and 7; reviewed in Refs. 3 and 8). On the A3 side of this direct, host–pathogen interaction, initial efforts suggested that the Vif binding sites in A3F and A3G may be structurally distinct, occurring in the C-terminus of the former and in the N-terminus of the latter at unrelated residues.[9], [10], [11], [12], [13], [14], [15] However, independent studies indicated that these single amino acid determinants of Vif recognition typically occur within a common region of a given susceptible A3 deaminase domain centered on the predicted α4 helix, suggesting that a conserved structural determinant may be targeted by Vif (DPD128–130 in A3G,[9], [10], [11], [12], [14], [15] E324 and E289 in A3F,[16], [17] and D/E121 in A3H,18 as well as multiple residues of a distinct region recently described in A3C, A3D, and A3F19).
Less is known about the downstream Ub acceptor sites targeted for polyUb during the degradation process. Iwatani et al. have previously reported that four lysines in the C-terminus of A3G are the Ub acceptor sites required for the Vif-mediated degradation of this enzyme.20 Combined with the aforementioned studies localizing Vif interaction to the N-terminal half of A3G, this led to the proposal of an antiparallel model of A3 binding to the Vif–E3 Ub ligase complex wherein Vif interacts with one deaminase domain, thereby orienting the second domain for polyUb by an activated E2–Ub conjugate at the opposite end of the CUL5 scaffold.
To test this model and potentially enhance our understanding both of A3 binding to the Vif–E3 Ub ligase complex and of how these substrate acceptor sites might be utilized to block the degradation of A3 proteins, we set out to define the sites of polyUb in A3F. Consistent with results previously reported by Iwatani et al., we find that internal lysines are the dominant Ub acceptor sites in both A3F and A3G. Analysis of the specific residues available for functional polyUb, however, reveals that these are dispersed throughout both domains of A3F at lysine residues clustered on one side of predicted structural models of the A3F N-terminal and C-terminal deaminase domains, opposite the Vif interaction site in the latter case. Furthermore, mutation of the lysine residues determined by Iwatani et al. to be the sites of polyUb in A3G confers only partial Vif resistance. Consistent with these genetic data, mass spectrometric analysis demonstrates Vif-dependent Ub modification of at least 6 sites in A3F and 10 sites in A3G localized to both the N- and C-terminal deaminase domains of each. We conclude, therefore, that the lysine residues available for Vif-dependent polyUb in human A3 proteins are diverse and therefore unlikely to be leveraged by novel therapeutics. To explain this flexibility, we further propose an alternative model of A3 binding to the Vif–E3 Ub ligase complex.
Section snippets
The sites of Vif-mediated polyUb in A3F are distributed throughout the protein
The lysine codons in A3F cluster into three linear groups separated by unique EcoRI and BamHI sites. We first used serial mutagenesis to convert each of these groups from K to R. Next, we joined the three regions together to make a panel of K-to-R mutants including a derivative completely devoid of lysines, A3F-19KR, and tested the restriction activity and Vif susceptibility of the resultant proteins in a single cycle of replication (Fig. 1a).
The restriction activity of all A3F mutants was
Discussion
We present here genetic and biophysical evidence for the polyUb of multiple internal lysines distributed throughout both the N- and C-termini of A3F and A3G (Fig. 1, Fig. 2, Fig. 3). The flexibility of the Vif–E3 ligase complex to promote the polyUb of A3 proteins at a wide range of acceptor sites is consistent with an active sampling of A3 lysines by the associated E2–Ub conjugate. Such a sampling model is further consistent with the observation that single R-to-K reversions in A3F-19KR
Plasmids
Wild-type A3F and A3G coding sequences are identical with those found in GenBank entries NM_145298 and NM_021822NM_145298NM_021822 and previously reported.41 A3 proteins were expressed from pcDNA3.1-derived vectors with a C-terminal V5 tag;41 in all V5 constructs except the Vif interaction mutants A3F QE323-324EK and A3G D128K, however, the inherent tag lysine was mutated to arginine to prevent artifactual tag Ub such as that previously described.20 K-to-R mutants were made by sequential
Acknowledgements
We thank A. K. Bielinsky, K. Walters, and the University of Minnesota Statistical Consulting Service for helpful discussion, and Y.-H. Zheng and the AIDS Research and Reference Reagent Program for materials. This research was funded by the National Institute of Allergy and Infectious Diseases (R01 AI064046 to R.S.H. and P01 AI090935 to N.J.K.) and the National Institute of General Medical Sciences (P01 GM091743 to R.S.H. and P50 GM082250 and P50 GM081879 to N.J.K.). J.S.A. was supported in part
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Characterization of an A3G-VifHIV-1-CRL5-CBFβ Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host-Pathogen Complexes
2021, Molecular and Cellular ProteomicsCitation Excerpt :Although the A3G-Vif interaction interface has been characterized in multiple mutagenesis experiments (see supplemental Tables S9 and S10 for extensive review; summarized in supplemental Fig. S13), our modeling combines these biochemical and genetically identified residues with cross-link data to describe the heptameric A3G-Vif-CRL5-CBFβ. The A3G-Vif interface includes regions that have been predicted to be part of the interface by multiple mutagenesis experiments (i.e., A3G residues 126–132, loops α1-β1, β2-α2, and β4-α4; and Vif 40–45 and 22–26) (21, 41, 93), as well as regions that have not previously predicted (e.g., A3G: 30–35, 55–66, 92–101, and 188–195). Importantly, we obtained a model ensemble that satisfies all the input information, including 99% of the input cross-links and the distances implied by the two residue–protein proximity restraints.
ARIH2 Is a Vif-Dependent Regulator of CUL5-Mediated APOBEC3G Degradation in HIV Infection
2019, Cell Host and MicrobeCitation Excerpt :Our in vitro data suggest that this effect is driven by a tag-team mechanism in which ARIH2 together with UBE2L3 act as initiators, priming A3G and A3F bound to neddylated CUL5Vif/CBFß with monoubiquitination, followed by chain elongation through UBE2R1 (Figures 5D, 5E, S5C, and S5D), a mechanism that is analogous to what was described for ARIH1 (Dove et al., 2017; Scott et al., 2016). Combined with the mapping of Ub sites on in vitro ubiquitinated A3G, which recapitulate previous sites detected in cells, (Figure 5F) (Albin et al., 2013) our results strongly suggest that this mechanism also leads to polyubiquitination of A3G in vivo. Beyond targeting APOBEC3 proteins during HIV infection, we further provided evidence that ARIH2 also tag-teams with other CRL5s to accelerate polyubiquitination of substrates, including CKB (Figures 7 and S3).
Role of co-expressed APOBEC3F and APOBEC3G in inducing HIV-1 drug resistance
2019, HeliyonCitation Excerpt :The HIV-1 protein Vif mediates the proteasomal degradation of several human A3 enzymes that are expressed in CD4 + T cells by interacting with several host factors to form a Cullin 5 E3 ubiquitin ligase assembly [18, 19, 20, 21, 22]. This results in the polyubiquitination and degradation of A3 enzymes [23, 24, 25, 26, 27, 28]. Without the action of Vif, higher numbers of A3 enzymes would bind HIV-1 genomic RNA or cellular RNA that is also bound by HIV-1 Gag and be encapsidated into virions [29, 30, 31].
Differential Contributions of Ubiquitin-Modified APOBEC3G Lysine Residues to HIV-1 Vif-Induced Degradation
2016, Journal of Molecular BiologyA computational analysis of the structural determinants of APOBEC3's catalytic activity and vulnerability to HIV-1 Vif
2014, VirologyCitation Excerpt :Recently determined structures of some APOBEC3 proteins enabled generating reliable models of the remaining APOBEC3 domains using the most homologous domains as templates. Although there have been previous efforts modeling APOBEC3 protein structures (Albin et al., 2010, 2013; Aydin et al., 2014; Belanger et al., 2013; Bulliard et al., 2009, 2011; Chelico et al., 2006; Chen et al., 2007; Desimmie et al., 2014; Harjes et al., 2009; Lackey et al., 2012; Lavens et al., 2010; Shirakawa et al., 2008; Stauch et al., 2009; Stenglein et al., 2008; Zhang et al., 2008; Zhen et al., 2010, 2012), a comprehensive and comparative analysis encompassing all human A3 domains has not been reported. Our analysis focuses on comparing the key characteristics relevant to the primary biological functions of the APOBEC3s.