Antiviral Factor APOBEC3G Research Articles

Structural insights into RNA bridging between HIV-1 Vif and antiviral factor APOBEC3G

Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV’s counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact. This establishes the biological significance of an RNA ligand. Further assessment of interactions between A3G, Vif, and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification, suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction.

Structural basis of sequence-specific RNA recognition by the antiviral factor APOBEC3G

An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the mechanism of APOBEC3G capturing viral RNA is unknown. Here, we report crystal structures of a primate APOBEC3G bound to different types of RNAs, revealing that APOBEC3G specifically recognizes unpaired 5’-AA-3’ dinucleotides, and to a lesser extent, 5’-GA-3’ dinucleotides. APOBEC3G binds to the common 3’A in the AA/GA motifs using an aromatic/hydrophobic pocket in the non-catalytic domain. It binds to the 5’A or 5’G in the AA/GA motifs using an aromatic/hydrophobic groove conformed between the non-catalytic and catalytic domains. APOBEC3G RNA binding property is distinct from that of the HIV nucleocapsid protein recognizing unpaired guanosines. Our findings suggest that the sequence-specific RNA recognition is critical for APOBEC3G virion packaging and restricting HIV infectivity.

Activities of endogenous APOBEC3s and uracil-DNA-glycosylase affect the hypermutation frequency of hepatitis B virus cccDNA.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) plays a key role in the persistence of viral infection. We have previously shown that overexpression of an antiviral factor APOBEC3G (A3G) induces hypermutation in duck HBV (DHBV) cccDNA, whereas uracil-DNA-glycosylase (UNG) reduces these mutations. In this study, using cell-culture systems, we examined whether endogenous A3s and UNG affect HBV cccDNA mutation frequency. IFNγ stimulation induced a significant increase in endogenous A3G expression and cccDNA hypermutation. UNG inhibition enhanced the IFNγ-mediated hypermutation frequency. Transfection of reconstructed cccDNA revealed that this enhanced hypermutation caused a reduction in viral replication. These results suggest that the balance of endogenous A3s and UNG activities affects HBV cccDNA mutation and replication competency.

HIV-1 adaptation studies reveal a novel Env-mediated homeostasis mechanism for evading lethal hypermutation by APOBEC3G.

HIV-1 replication normally requires Vif-mediated neutralization of APOBEC3 antiviral enzymes. Viruses lacking Vif succumb to deamination-dependent and -independent restriction processes. Here, HIV-1 adaptation studies were leveraged to ask whether viruses with an irreparable vif deletion could develop resistance to restrictive levels of APOBEC3G. Several resistant viruses were recovered with multiple amino acid substitutions in Env, and these changes alone are sufficient to protect Vif-null viruses from APOBEC3G-dependent restriction in T cell lines. Env adaptations cause decreased fusogenicity, which results in higher levels of Gag-Pol packaging. Increased concentrations of packaged Pol in turn enable faster virus DNA replication and protection from APOBEC3G-mediated hypermutation of viral replication intermediates. Taken together, these studies reveal that a moderate decrease in one essential viral activity, namely Env-mediated fusogenicity, enables the virus to change other activities, here, Gag-Pol packaging during particle production, and thereby escape restriction by the antiviral factor APOBEC3G. We propose a new paradigm in which alterations in viral homeostasis, through compensatory small changes, constitute a general mechanism used by HIV-1 and other viral pathogens to escape innate antiviral responses and other inhibitions including antiviral drugs.

Effect of vaginal immunization with HIVgp140 and HSP70 on HIV-1 replication and innate and T cell adaptive immunity in women.

The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4(+) T cells compared with preimmunization CD4(+) T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4(+) T cells. Indeed, a significant inverse correlation between the proportion of CCR5(+) T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4(+) and CD8(+) T cell proliferative responses were significantly increased and CD4(+) T cells showed a trend to have an inverse correlation with the viral load (r = -0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at ClinicalTrials.gov under registration no. NCT01285141.) Importance: Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4(+) T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4(+) T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4(+) T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4(+) and CD8(+) T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4(+) T cells and the stimulation of CD4(+) or CD8(+) T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4(+) and CD8(+) T cell proliferative responses.

Viral Infectivity Factor: A Novel Therapeutic Strategy to Block HIV-1 Replication

The HIV-1 viral infectivity factor (Vif), one of the six accessory proteins, is essential for viral replication and pathogenesis. Its main function is to form Vif-Cullin5-ElonginBC complex and then is able to ubiquitinate and degrade the human anti-viral factor APOBEC3G, which markedly enhances the virus infectivity. Delete Vif leads to the loss of the infectivity of HIV-1; therefore, Vif is a potentially new attractive target for therapeutic intervention in AIDS. This review describes the structure, function and especially inhibitors of HIV-1 Vif.

The antiviral factor APOBEC3G enhances the recognition of HIV-infected primary T cells by natural killer cells

New from NPG: The antiviral factor APOBEC3G enhances the recognition of HIV-infected primary T cells by natural killer cells

Damage control: how HIV survives the editor APOBEC3G

The antiviral factor APOBEC3G upregulates the expression of ligands for the activating receptor NKG2D via DNA damage induced by the viral protein Vpr in cells infected with human immunodeficiency virus. The virus overcomes greater susceptibility to natural killer cell–mediated lysis by targeting APOBEC3G for degradation.

The antiviral factor APOBEC3G enhances the recognition of HIV-infected primary T cells by natural killer cells

APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits HIV replication by deaminating cytidine residues to uridine. This causes G-to-A hypermutation in the opposite strand and results in viral inactivation. HIV counteracts A3G through the activity of viral infectivity factor (Vif), which promotes A3G degradation. We report that viral protein R (Vpr), which interacts with a uracil glycosylase, also counteracts A3G by reducing uridine incorporation. However, this process results in activation of the DNA damage response pathway and expression of NK cell activating ligands. Our results reveal that pathogen-induced cytidine deamination and the DNA damage response to viral-mediated repair of uridine incorporation enhance recognition of HIV-infected cells by NK cells.

Mutation Vif-22H, which allows HIV-1 to use the APOBEC3G hypermutation to develop resistance, could appear more quickly in certain non-B variants

Sir, HIV-1 Vif protein counteracts the lethal hypermutation produced by the cellular antiviral factor APOBEC3G (A3G) in HIV-1. This effect consists of a cytidine deamination of the HIV-1 cDNA that results in guanosine to adenosine (G-to-A) substitutions in the proviral sequences in a GG-to-GA dinucleotide context. However, in defective Vif alleles these G-to-A substitutions could benefit the virus, promoting sequence diversification even in drug resistance sites, and therefore accelerate the evolution of resistance and immune escape. It would represent a new mechanism of viral escape from antiretrovirals and a suitable target for drug development. In this context, the mutation Vif-K22H (located in the Vif–A3G interaction domain) has been recently identified as a key mutation to trigger this mechanism in subjects under therapy failure. However, all the described Vif-K22H samples were subtype B. Most studies are based on this subtype because it is prevalent in developed regions, despite the worldwide predominance of HIV-1 non-B variants, a term that includes the remaining subtypes and inter-subtype recombinants. Here we analyse the presence of Vif-K22H in the most prevalent HIV-1 non-B subtypes and recombinants, which, up to now, is still unknown. All HIV-1 vif sequences (n1⁄42895) ascribed to pure subtypes and recombinants CRF01_AE and CRF02_AG were downloaded from the Los Alamos National Laboratory Database (LANL-DB) in March 2010: 143 A1; 5 A2; 1690 B; 546 C; 91 D; 36 F1; 4 F2; 30 G; 4 H; 3 J; 2 K; 242 CRF01_AE; and 99 CRF02_AG. Nucleotide and amino acid variability in Vif-22 across all HIV-1 studied subtypes and recombinants was identified and compared (Table 1). Table 1. Amino acid and nucleotide variability at position 22 among the 2895 vif sequences deposited in the LANL-DB according to HIV-1 clade

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti–human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1–specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

A Hydrodynamic Analysis of APOBEC3G Reveals a Monomer−Dimer−Tetramer Self-Association That Has Implications for Anti-HIV Function

The innate antiviral factor APOBEC3G (A3G) possesses RNA binding activity and deaminates HIV-1 DNA. High-molecular mass forms of A3G can be isolated from a variety of cell types but exhibit limited deaminase activity relative to low-molecular mass species prepared under RNA-depleted conditions. To investigate the fundamental oligomeric state and shape of A3G, we conducted sedimentation velocity analyses of the pure enzyme under RNA-deficient conditions. The results reveal a predominant dimer in equilibrium with minor monomeric and tetrameric species. Hydrodynamic modeling of the dimer supports an extended cylindrical shape that assembles into an elongated tetramer. Overall, the results provide physical restraints for the A3G quaternary structure that have implications for modulating antiviral function.

Endogenous factors enhance HIV infection of tissue naive CD4 T cells by stimulating high molecular mass APOBEC3G complex formation

Human immunodeficiency virus (HIV) can infect resting CD4 T cells residing in lymphoid tissues but not those circulating in peripheral blood. The molecular mechanisms producing this difference remain unknown. We explored the potential role of the tissue microenvironment and its influence on the action of the antiviral factor APOBEC3G (A3G) in regulating permissivity to HIV infection. We found that endogenous IL-2 and -15 play a key role in rendering resident naive CD4 T cells susceptible to HIV infection. Infection of memory CD4 T cells also requires endogenous soluble factors, but not IL-2 or -15. A3G is found in a high molecular mass complex in HIV infection–permissive, tissue-resident naive CD4 T cells but resides in a low molecular mass form in nonpermissive, blood-derived naive CD4 T cells. Upon treatment with endogenous soluble factors, these cells become permissive for HIV infection, as low molecular mass A3G is induced to assemble into high molecular mass complexes. These findings suggest that in lymphoid tissues, endogenous soluble factors, likely including IL-2 and -15 and others, stimulate the formation of high molecular mass A3G complexes in tissue-resident naive CD4 T cells, thereby relieving the potent postentry restriction block for HIV infection conferred by low molecular mass A3G.

Cooperative and Specific Binding of Vif to the 5′ Region of HIV-1 Genomic RNA

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.

Regulation of Apobec3F and Human Immunodeficiency Virus Type 1 Vif by Vif-Cul5-ElonB/C E3 Ubiquitin Ligase

The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully understood. Here, we demonstrate that while h-A3F, like h-A3G, was able to suppress primate lentiviruses other than HIV-1 (simian immunodeficiency virus from African green monkeys [SIVagm] and Rhesus macaques [SIVmac]), the interaction between Vif proteins and h-A3F appeared to differ from that with h-A3G. H-A3F showed no change in its species specificity against HIV-1 or SIVagm Vif when a negatively charged amino acid was replaced with a lysine at position 128, a residue critical for h-A3G recognition by HIV-1 Vif. However, HIV-1 Vif, but not SIVagm Vif, was able to bind h-A3F and induce its polyubiquitination and degradation through the Cul5-containing E3 ubiquitin ligase. Interference with Cul5-E3 ligase function by depletion of Cul5, through RNA interference or overexpression of Cul5 mutants, blocked the ability of HIV-1 Vif to suppress h-A3F. A BC-box mutant of HIV-1 Vif that failed to recruit Cul5-E3 ligase but was still able to interact with h-A3F failed to suppress h-A3F. Interestingly, interference with Cul5-E3 ligase function or overexpression of h-A3F or h-A3G also increased the stability of HIV-1 Vif, suggesting that like the substrate molecules h-A3F and h-A3G, the substrate receptor protein Vif is itself also regulated by Cul5-E3 ligase. Our results indicate that Cul5-E3 ligase appears to be a common pathway hijacked by HIV-1 Vif to defeat both h-A3F and h-A3G. Developing inhibitors to disrupt the interaction between Vif and Cul5-E3 ligase could be therapeutically useful, allowing multiple host antiviral factors to suppress HIV-1.

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.