Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd''s deamination reactions within a 5′-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5′-CCC substrate over 5′-CCU substrate, which explains why A3G displays almost no processivity within a 5′-CCC motif. In addition, A3Gctd''s shortest substrate sequence was found to be a pentanucleotide containing 5′-CCC flanked on both sides by a single nucleotide. A3Gctd as well as full-length A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo.     

Signals in APOBEC3F N-terminal and C-terminal deaminase domains each contribute to encapsidation in HIV-1 virions and are both required for HIV-1 restriction

Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) inhibit human immunodeficiency virus type-1 (HIV-1) replication. In the absence of HIV-1 Vif, A3F and/or A3G are incorporated into assembling virions and exert antiviral functions in subsequently infected target cells. Encapsidation of A3F or A3G within the protease-matured virion core following their incorporation into virions is hypothesized to be important for the antiviral function of these proteins. In this report, we demonstrated that A3F was quantitatively encapsidated in the mature virion core. In distinct contrast, A3G was distributed both within and outside of the virion core. Analysis of a series of A3F-A3G chimeras comprised of exchanged N- and C-terminal deaminase domains identified a 14 amino acid segment in the A3F C-terminal deaminase domain that contributed to preferential encapsidation and anti-HIV activity. Amino acid residue L306 in this C-terminal segment was determined to be necessary, but not sufficient, for these effects. Amino acid residue W126 in the N-terminal deaminase domain was determined also to contribute to preferential encapsidation and antiviral activity of A3F. Analysis of the A3F (W126A L306A) double mutant revealed that both residues are required for full anti-HIV function. The results reported here advance our understanding of the mechanisms of A3F virion encapsidation and antiviral function and may lead to innovative strategies to inhibit HIV-1 replication.     

Differential Anti-APOBEC3G Activity of HIV-1 Vif Proteins Derived from Different Subtypes

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.     

Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif

HIV-1 Vif assembles the Cul5-EloB/C E3 ubiquitin ligase to induce proteasomal degradation of the cellular antiviral APOBEC3 proteins. Detailed structural studies have confirmed critical functional domains in Vif that we have previously identified as important for the interaction of EloB/C, Cul5, and CBFβ. However, the mechanism by which Vif recognizes substrates remains poorly understood. Specific regions of Vif have been identified as being responsible for binding and depleting APOBEC3G and APOBEC3F. Interestingly, we have now identified distinct yet overlapping domains that are required for HIV-1 Vif-mediated G2/M-phase cell cycle arrest and APOBEC3H degradation, but not for the inactivation of APOBEC3G or APOBEC3F. Surprisingly, Vif molecules from primary HIV-1 variants that caused G2/M arrest were unable to inactivate APOBEC3H; on the other hand, HIV-1 Vif variants that could inactivate APOBEC3H were unable to induce G2/M arrest. All of these Vif variants still maintained the ability to inactivate APOBEC3G/F. Thus, primary HIV-1 variants have evolved to possess distinct functional activities that allow them to suppress APOBEC3H or cause G2 cell cycle arrest, using mutually exclusive interface domains. APOBEC3H depletion and G2 arrest are apparently evolutionary selected features that cannot co-exist on a single Vif molecule. The existence and persistence of both types of HIV-1 Vif variant suggests the importance of APOBEC3H suppression and cell cycle regulation for HIV-1''s survival in vivo.     

Enzymatically active APOBEC3G is required for efficient inhibition of human immunodeficiency virus type 1

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif-defective human immunodeficiency virus type 1 requires catalytically active APO3G.     

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.     

APOBEC3D and APOBEC3F Potently Promote HIV-1 Diversification and Evolution in Humanized Mouse Model

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.     

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant Nature of APOBEC3G

Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10⁻² to 10⁻⁵ in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR (∼10⁻⁴ to 10⁻⁵). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Δvif efficiently.     

HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.     

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.     

APOBEC3G Restricts HIV-1 to a Greater Extent than APOBEC3F and APOBEC3DE in Human Primary CD4+ T Cells and Macrophages

APOBEC3 proteins inhibit HIV-1 replication in experimental systems and induce hypermutation in infected patients; however, the relative contributions of several APOBEC3 proteins to restriction of HIV-1 replication in the absence of the viral Vif protein in human primary CD4⁺ T cells and macrophages are unknown. We observed significant inhibition of HIV-1Δvif produced in 293T cells in the presence of APOBEC3DE (A3DE), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H haplotype II (A3H HapII) but not APOBEC3B (A3B), APOBEC3C (A3C), or APOBEC3H haplotype I (A3H HapI). Our previous studies showed that Vif amino acids Y⁴⁰RHHY⁴⁴ are important for inducing proteasomal degradation of A3G, whereas amino acids ¹⁴DRMR¹⁷ are important for degradation of A3F and A3DE. Here, we introduced substitution mutations of ⁴⁰YRHHY⁴⁴ and ¹⁴DRMR¹⁷ in replication-competent HIV-1 to generate vif mutants NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 to compare the antiviral activity of A3G to the combined antiviral activity of A3F and A3DE in activated CD4⁺ T cells and macrophages. During the first 15 days (round 1), in which multiple cycles of viral replication occurred, both the NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutants replicated in activated CD4⁺ T cells and macrophages, and only the NL4-3 YRHHY>A5 mutant showed a 2- to 4-day delay in replication compared to the wild type. During the subsequent 27 days (round 2) of cultures initiated with peak virus obtained from round 1, the NL4-3 YRHHY>A5 mutant exhibited a longer, 8- to 10-day delay and the NL4-3 DRMR>A4 mutant exhibited a 2- to 6-day delay in replication compared to the wild type. The NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutant proviruses displayed G-to-A hypermutations primarily in GG and GA dinucleotides as expected of A3G- and A3F- or A3DE-mediated deamination, respectively. We conclude that A3G exerts a greater restriction effect on HIV-1 than A3F and A3DE.     

The Resistance of Human APOBEC3H to HIV-1 NL4-3 Molecular Clone Is Determined by a Single Amino Acid in Vif

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.     

Evolutionarily conserved and non-conserved retrovirus restriction activities of artiodactyl APOBEC3F proteins

The APOBEC3 proteins are unique to mammals. Many inhibit retrovirus infection through a cDNA cytosine deamination mechanism. HIV-1 neutralizes this host defense through Vif, which triggers APOBEC3 ubiquitination and degradation. Here, we report an APOBEC3F-like, double deaminase domain protein from three artiodactyls: cattle, pigs and sheep. Like their human counterparts, APOBEC3F and APOBEC3G, the artiodactyl APOBEC3F proteins are DNA cytosine deaminases that locate predominantly to the cytosol and can inhibit the replication of HIV-1 and MLV. Retrovirus restriction is attributable to deaminase-dependent and -independent mechanisms, as deaminase-defective mutants retain significant anti-retroviral activity. However, unlike human APOBEC3F and APOBEC3G, the artiodactyl APOBEC3F proteins have an active N-terminal DNA cytosine deaminase domain, which elicits a broader dinucleotide deamination preference, and they are resistant to HIV-1 Vif. These data indicate that DNA cytosine deamination; sub-cellular localization and retrovirus restriction activities are conserved in mammals, whereas active site location, local mutational preferences and Vif susceptibility are not. Together, these studies indicate that some properties of the mammal-specific, APOBEC3-dependent retroelement restriction system are necessary and conserved, but others are simultaneously modular and highly adaptable.