Distinct viral determinants for the packaging of human cytidine deaminases APOBEC3G and APOBEC3C

Human APOBEC3G and other APOBEC3 cytidine deaminases inhibit a variety of retroviruses, including Vif-deficient HIV-1. These host proteins are packaged into viral particles and inhibit the replication of virus in new target cells. A3G and A3F are known to be efficiently packaged into HIV-1 virions by binding to 7SL RNA through the Gag NC domain; however, the packaging mechanisms of other APOBEC3 proteins are poorly defined. We have now demonstrated that APOBEC3C (A3C) can be efficiently packaged into HIV-1 virions that are deficient for viral genomic RNA. Inhibition of the encapsidation of 7SL RNA into HIV-1 virions blocked the packaging of A3G, but not A3C. While the NC domain is required for efficient packaging of A3G, deletion of this domain had little effect on A3C packaging into HIV-1 Gag particles. A3C interacted with HIV-1 Gag which was MA domain-dependent and RNA-dependent. Deletion of the MA domain of HIV-1 Gag inhibited A3C but not A3G packaging into HIV-1 Gag particles. Thus, A3G and A3C have evolved to use distinct mechanisms for targeting retroviruses.     

胞嘧啶脱氨酶APOBEC3G可能通过G→A突变抑制乙型肝炎病毒的复制

目的研究胞嘧啶脱氨酶APOBEC3G(A3G)对HBV复制的影响及作用机制。方法利用脂质体介导A3G和HBV的真核表达质粒瞬时共转染人肝癌细胞株HepG2,以空载体pcDNA3.1与HBV真核表达质粒共转染为对照,同时转染含增强型绿色荧光蛋白(EGFP)基因的质粒载体以判定转染效率。共转染两天后用荧光定量PCR方法定量细胞内核壳体(core)相关HBV DNA水平,用Western blot检测细胞内A3G和HBcAg的表达,并对细胞内core相关HBV DNA X基因进行PCR扩增、T-A克隆和测序,分析X基因中的碱基突变。结果经EGFP判定的转染效率平均为29%。共转染0.5μgA3G和0.5μgHBV的HepG2细胞内core相关HBV DNA量平均下降为对照的8.9%,共转染2μgA3G和0.5μgHBV的平均下降为对照的0.6%。共转染A3G和HBV表达质粒后,HepG2细胞内HBV的X基因中发生G→A突变的数目明显增多,33个克隆中有9个克隆检测到了16～37个G→A突变,突变总数达254个,而对照的33个克隆中仅有2个克隆各检测到2个G→A突变。进一步的分析发现,共转染A3G后发生的G→A突变大多集中于几个热点区域,突变靶点常在GG二核苷酸中的第一个G上。结论胞嘧啶脱氨酶APOBEC3G可能通过诱导。HBV DNA的X基因发生G→A超突变,抑制HBV在人肝癌细胞HepG2中的复制。     

Functional analysis of the two cytidine deaminase domains in APOBEC3G

Human APOBEC3G (hA3G), a cytidine deaminase with two cytidine deaminase domains (CDs), has been identified as an anti-HIV-1 host factor. Although the two CDs of hA3G have been extensively characterized, there is still debate on the role of the CDs in the biological function of hA3G. In this work, we constructed three hA3G mutants CD1-1, CD2-2 and CD2-1, which contain duplicate CD1 domain, duplicate CD2 domain and position switched CD domain respectively, and investigated the effect of CD domain replacement or switch upon virion encapsidation, Vif-mediated degradation, deamination and antiviral activity of hA3G. The results showed that the two CD domains were functionally equivalent in virion encapsidation and the interaction with HIV-1 Vif of hA3G, whereas CD domain switch or replacement greatly affected the sensitivity to Vif induced degradation, editing and antiviral activity of hA3G. Although the CD2 domain was shown to possess the deamination activity, CD2-2 incorporated efficiently into HIV-1 was unable to mutate viral cDNA, suggesting that CD1 also involved in the enzymatic function. Interestingly, CD2-1 retained considerable deamination activity with a different sequence preference. Taken together, our results suggest that CD domain may play a structural role in virion encapsidation and Vif-mediated degradation of hA3G, and coordination of the two CD domains is required for its editing and antiviral activity.     

HIV-1 Vif promotes the formation of high molecular mass APOBEC3G complexes

HIV-1 Vif inhibits the antiviral activity of APOBEC3G (APO3G) by inducing proteasomal degradation. Here, we studied the effects of Vif on APO3G in vitro. In this system, Vif did not cause APO3G degradation. Instead, Vif induced changes in APO3G that affected immunoprecipitation of the native protein. This effect required wt Vif and was reversed by heat denaturation of APO3G. Sucrose gradient analysis demonstrated that wt Vif induced the gradual transition of APO3G translated in vitro or expressed in HeLa cells from a low molecular mass conformation to puromycin-sensitive high molecular mass (HMM) complexes. In the absence of Vif or the presence of biologically inactive Vif APO3G failed to form HMM complexes. Our results expose a novel function of Vif that promotes the assembly of APO3G into presumably packaging-incompetent HMM complexes and may explain how Vif can overcome the APO3G-imposed block to HIV replication under conditions of no or inefficient APO3G degradation.     

Inhibition of LINE-1 and Alu retrotransposition by exosomes encapsidating APOBEC3G and APOBEC3F

Human cytidine deaminases, including APOBEC3G (A3G) and A3F, are part of a cellular defense system against retroviruses and retroelements including non-LTR retrotransposons LINE-1 (L1) and Alu. Expression of cellular A3 proteins is sufficient for inhibition of L1 and Alu retrotransposition, but the effect of A3 proteins transferred in exosomes on retroelement mobilization is unknown. Here, we demonstrate for the first time that exosomes secreted by CD4⁺H9 T cells and mature monocyte-derived dendritic cells encapsidate A3G and A3F and inhibit L1 and Alu retrotransposition. A3G is the major contributor to the inhibitory activity of exosomes, however, the contribution of A3F in H9 exosomes cannot be excluded. Additionally, we show that exosomes encapsidate mRNAs coding for A3 proteins. A3G mRNA, and less so A3F, was enriched in exosomes secreted by H9 cells. Exosomal A3G mRNA was functional in vitro. Whether exosomes inhibit retrotransposons in vivo requires further investigation.     

Comparative analysis of the antiretroviral activity of APOBEC3G and APOBEC3F from primates

APOBEC3G and APOBEC3F exhibit antiretroviral activity primarily as a consequence of their ability to deaminate cytidines in retroviral DNA. Here, we compare the properties of APOBEC3F and APOBEC3G from human, macaque, and African green monkey (AGM). While all APOBEC proteins tested exhibited anti-HIV-1 activity, human APOBEC3F was, surprisingly, 10- to 50-fold less potent than human APOBEC3G. However, similar discrepancies in antiviral potency were not found when pairs of proteins from macaque and AGM were compared. Intrinsic differences in the ability of each APOBEC protein to induce hypermutation, rather than differences in packaging efficiency, partially accounted for variable antiretroviral activity. Each of four primate lentivirus Vif proteins reduced human and AGM APOBEC3F expression and antiviral activity, but all were only partially effective and species-specific effects were relatively minor. Overall, highly efficient and species-specific neutralization of APOBEC3G, and less efficient neutralization of APOBEC3F, appears to be a general property of Vif proteins.     

Expression of APOBEC3G in kidney cells

The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a member of the APOBEC family possessing DNA mutator activity through cytosine deamination, is reported to play an important role in host defense against infections such as those of hepatitis B virus and human immunodeficiency virus. Here, we examined the expression of APOBEC3G in human kidney cells to better understand its biological role against infection. APOBEC3G was immunohistochemically detectable in kidney mesangial cells and also to some extent in kidney epithelial tubular cells. In addition, overexpression of APOBEC3G was shown in renal carcinoma tissues and cell lines. APOBEC3G expression was upregulated by inflammatory cytokines, such as interferon, interleukin-6, and tumor necrosis factor. These results may provide new insight into the role of APOBEC3G in host defense against viral infection and cancer.     

IFN-α诱导HuH7细胞固有免疫分子APOBEC3G的表达

目的了解IFN-α对HuH7细胞固有免疫分子APOBEC3G表达的影响及其机制。方法HuH7细胞给予不同浓度的IFN-α(0 U/ml、100U/ml、400U/ml、800U/ml and 1200U/ml)刺激,10h后提取细胞总RNA,RT-PCR和实时定量RT-PCR检测HuH7细胞内APOBEC3G的mRNA水平变化,Western blot分析APOBEC3G蛋白水平的表达;分别构建不同长度的含有IRF-E(IFN regulatory factor element)位点的APOBEC3G起始密码上游序列报告质粒、不含IRF-E和IRF-E位点突变的虫荧光素酶报告质粒,报告基因分析IFN-α刺激后APOBEC3G起始密码上游IPF-E位点对APOBEC3G表达的影响。结果IFN-α上调HuH7细胞APOBEC3G mRNA和蛋白水平的表达,并具有剂量依赖的效应。序列分析发现APOBEC3G起始密码上游的-298～-283和-57～-47位碱基分别存在IRF-E和ISRE(IFN stimulated re- sponse element)序列。报告基因分析结果显示,干扰素使含有IRF-E序列的APOBEC3G启动子报告质粒的虫荧光素酶的活性增加6～8倍,而不含IRF-E序列和IRF-E位点突变的报告质粒的虫荧光素酶活性在干扰素刺激后几乎没有变化。结论IFN-α可通过IRF-E位点上调APOBEC3G mRNA和蛋白水平的表达,从而发挥抗病毒效应。     

The HIV-1 Vif PPLP motif is necessary for human APOBEC3G binding and degradation

The HIV-1 virion infectivity factor (Vif) is required during viral replication to inactivate the host cell anti-viral factor, APOBEC3G (A3G). Vif binds A3G and a Cullin5-ElonginBC E3 ubiquitin ligase complex which results in the proteasomal degradation of A3G. The Vif PPLP motif (amino acids 161-164) is essential for normal Vif function because mutations in this motif reduce the infectivity of virions produced in T-cells. In this report, we demonstrate that mutation of the Vif PPLP motif reduces Vif binding to A3G without affecting its interaction with ElonginC and Cullin5. We demonstrate that the failure of the Vif mutant to bind A3G resulted in A3G incorporation into assembling virions with loss of viral infectivity.     

Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation

HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the DNA deaminase APOBEC3G by targeting it for proteasomal degradation. We report here that Vif targets APOBEC3G for degradation by forming an SCF-like E3 ubiquitin ligase containing Cullin 5 and Elongins B and C (Cul5-EloB-EloC) through a novel SOCS (suppressor of cytokine signaling)-box that binds EloC. Vif binding to EloC is negatively regulated by serine phosphorylation in the BC-box motif of the SOCS-box. Vif ubiquitination is promoted by Cul5 in vitro and in vivo, and requires an intact SOCS-box. Thus, autoubiquitination of Vif occurs within the assembled Vif-Cul5 complex, analogous to F-box proteins that are autoubiquitinated within their SCF (Skp1-Cullin-F-box) complex. These findings suggest mechanisms that regulate the assembly and activity of Cul5 E3 complexes through phosphorylation or autoubiquitination of the SOCS-box protein, and identify interactions between Vif and host cell proteins that may be therapeutic targets.     

APOBEC3G-independent reduction in virion infectivity during long-term HIV-1 replication in terminally differentiated macrophages

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. In the case of HIV, the antiviral activity of APO3G is counteracted by the viral Vif protein. Monocyte-derived macrophages (MDM) are terminally differentiated, non-dividing cells susceptible to HIV infection. Human MDM are known to express APO3G and HIV replication in these cells is dependent on Vif. Here we analyzed the correlation between HIV-1 replication and APO3G expression in MDM. Replication of wild type HIV-1 induced a gradual 4-5-fold reduction in APO3G expression. The efficiency of APO3G downregulation correlated with the efficiency of virus replication. Interestingly, despite downregulation of APO3G, the relative infectivity of viruses rapidly declined during the course of infection and was already reduced ∼ 90% prior to peak virus production. Cell-free virus preparations showed increased levels of a 41 kDa MA-CA processing intermediate. Sequence analysis around the MA-CA cleavage site and the protease and LTR regions did not reveal deaminase-induced hypermutation of the viral genome, suggesting that APO3G activity is not responsible for the incomplete Gag processing. Thus, the loss of infectivity of HIV-1 viruses produced from long-term infected primary macrophages is due to an APO3G-independent mechanism.     

The interferon-induced expression of APOBEC3G in human blood-brain barrier exerts a potent intrinsic immunity to block HIV-1 entry to central nervous system

In the human genome, the APOBEC3 gene has expanded into a tandem array of genes termed APOBEC3A-H. Several members of this family have potent anti-HIV-1 activity. Here we demonstrate that APOBEC-3B/3C/3F and -3G are expressed in all major cellular components of the CNS. Moreover, we show that both interferon-alpha (IFN-alpha) and IFN-gamma significantly enhance the expression of APOBEC-3G/3F and drastically inhibit HIV-1 replication in primary human brain microvascular endothelial cells (BMVECs), the major component of blood-brain barrier (BBB). As the viral inhibition can be neutralized by APOBEC3G-specific siRNA, APOBEC3G plays a key role to mediate the anti-HIV-1 activity of IFN-alpha and/or IFN-gamma. Our findings suggest that, in addition to the restriction at viral entry level, the restriction from APOBEC3 family could account for the low-level replication of HIV-1 in BMVECs. The manipulation of IFN-APOBEC3 signaling pathway could be a potent therapeutic strategy to prevent HIV invasion to central nervous system (CNS).     

Complementary function of the two catalytic domains of APOBEC3G

The HIV-1 viral accessory protein Vif prevents the encapsidation of the antiviral cellular cytidine deaminases APOBEC3F and APOBEC3G by inducing their proteasomal degradation. In the absence of Vif, APOBEC3G is encapsidated and blocks virus replication by deaminating cytosines of the viral cDNA. APOBEC3G encapsidation has been recently shown to depend on the viral nucleocapsid protein; however, the role of RNA remains unclear. Using APOBEC3G deletion and point mutants, we mapped the encapsidation determinant to the Zn(2+) coordination residues of the N-terminal catalytic domain (CD1). Notably, these residues were also required for RNA binding. Mutations in the two aromatic residues of CD1 but not CD2, which are conserved in cytidine deaminase core domains and are required for RNA binding, prevented encapsidation into HIV-1, HTLV-I and MLV. The Zn(2+) coordination residues of the C-terminal catalytic domain (CD2) were not required for encapsidation but were essential for cytidine deaminase activity and the antiviral effect. These findings suggest a model in which CD1 mediates encapsidation and RNA binding while CD2 mediates cytidine deaminase activity. Interestingly, HTLV-I was relatively resistant to the antiviral effects of encapsidated APOBEC3G.     

Analysis of Vif-induced APOBEC3G degradation using an alpha-complementation assay

Vif forms a complex with Elongin B/C, Cullin-5 and Rbx-1 to induce the polyubiquitination and proteasome-mediated degradation of human APOBEC3G (hA3G). These interactions serve as potential targets for anti-HIV-1 drug development. We have developed a cell culture-based assay to measure Vif-induced hA3G degradation. The assay is based on alpha-complementation, the ability of beta-galactosidase fragments to complement in trans. hA3G expressed with a fused alpha-peptide was enzymatically active, complemented a coexpressed omega-fragment and could be targeted for degradation by Vif. Vif reduced beta-galactosidase activity in the cell by 10-30-fold. The assay was validated by testing various hA3G and Vif point mutants. The assay accurately detected the effects of D128 in hA3G, and the BC box, Cul5 box and HCCH motifs of Vif. The results showed a strict association of Vif biological function with hA3G degradation. These findings support hA3G degradation as a requirement for Vif function. The Vif alpha-complementation assay may be a useful tool for the identification of Vif inhibitors.     

Function analysis of sequences in human APOBEC3G involved in Vif-mediated degradation

Human APOBEC3G (hA3G) has been identified as an anti-HIV cellular factor. As a counter measure, the HIV-1 protein Vif causes the degradation of hA3G by binding to it and directing it to the cellular proteasome. In this work, we have used hA3G deletion mutants to map the region in hA3G required for its degradation by Vif to hA3G amino acids 105-245, the linker region between the two zinc coordination motifs. A small fragment of hA3G containing only amino acids 105-245 will undergo Vif-induced degradation. However, while amino acids 105-156 of hA3G are required for Vif interaction with hA3G, they are not themselves sufficient for hA3G degradation, a process that further requires amino acids 157-245. While expression of hA3G fragments 1-156 or 157-384 (but not 246-384) can dominantly inhibit the Vif-mediated degradation of full-length hA3G, only the N-terminal fragment inhibits the Vif/hA3G interaction. Inhibition of hA3G degradation by the C-terminal hA3G fragment 157-384 appears to be related to its ability to prevent the polyubiquitination of hA3G induced by Vif, a process that is required for Vif-mediated proteosomal degradation of hA3G. Non-permissive cells stably expressing hA3G 1-156 or hA3G 157-384 are able to inhibit the replication of wild-type HIV-1, thereby verifying the inhibitory effect of these fragments upon Vif-mediated hA3G degradation and suggesting their potential in anti-HIV-1 therapy.     

Production of infectious virus and degradation of APOBEC3G are separable functional properties of human immunodeficiency virus type 1 Vif

HIV-1 Vif regulates viral infectivity by inhibiting the encapsidation of APOBEC3G (APO3G) through proteasomal degradation of the protein. Here we compared various Vif proteins for their ability to induce APO3G degradation and rescue viral infectivity. We found that Vif expressed from proviral vectors caused relatively inefficient degradation of APO3G in HeLa cells yet was very effective in inhibiting APO3G's antiviral activity. On the other hand, Vif expressed autonomously from a codon-optimized vector caused very efficient APO3G degradation and also effectively inhibited APO3G's antiviral effects. In contrast, a Vif chimera containing an N-terminal fluorescent tag efficiently induced APO3G degradation but was unable to restore viral infectivity. The lack of a direct correlation between APO3G degradation and rescue of viral infectivity suggests that these two properties of Vif are functionally separable. Our data imply that intracellular degradation of APO3G may not be the sole activity of Vif required for the production of infectious virions from APO3G-expressing cells.     

In-depth analysis of G-to-A hypermutation rate in HIV-1 env DNA induced by endogenous APOBEC3 proteins using massively parallel sequencing

Some APOBEC3 proteins cause G-to-A hypermutation in HIV-1 DNA when the accessory viral protein Vif is absent or non-functional. So far, cloning and sequencing has been performed to study G-to-A hypermutation. This is time-consuming and labour-intensive especially in the context of in vivo investigations where the number of hypermutated sequences can be very low. Thus, a massively parallel sequencing protocol has been developed for in-depth analysis of G-to-A hypermutation using the 454 pyrosequencing FLX system. Part of HIV-1 env was amplified and pyrosequenced after two rounds of infection in T cell lines and PBMCs using HIV-1 NL4-3Δvif. Specific criteria were applied to cope with major technical challenges: (1) the inclusion of hypermutated sequences, (2) the high genome diversity of HIV-1 env, and (3) the exclusion of sequences containing frameshift errors caused by pyrosequencing. In total, more than 140,000 sequences were obtained. 1.3-6.5% of guanines were mutated to adenine, most frequently in the GG dinucleotide context, the preferred deamination site of APOBEC3G. Non-G-to-A mutations occurred only in low frequencies (<0.6%). Single hypermutated sequences contained up to 24 G-to-A mutations. Overall, massively parallel sequencing is a very useful tool for in-depth analysis of G-to-A hypermutation in HIV-1 DNA induced by APOBEC3 proteins.     

Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif

While the human antiretroviral defense factors APOBEC3F and APOBEC3G are potent inhibitors of the replication of HIV-1 mutants lacking a functional vif gene, the Vif protein expressed by wild-type HIV-1 blocks the function of both host cell proteins. Here, we report that a third human protein, APOBEC3B, is able to suppress the infectivity of both Vif-deficient and wild-type HIV-1 with equal efficiency. APOBEC3B, which shows approximately 58% sequence identity to both APOBEC3F and APOBEC3G, shares the ability of these other human proteins to bind the nucleocapsid domain of HIV-1 Gag specifically and to thereby package into progeny virion particles. However, APOBEC3B differs from APOBEC3F and APOBEC3G in that it is unable to bind to HIV-1 Vif in co-expressing cells and is therefore efficiently packaged into HIV-1 virions regardless of Vif expression. Unfortunately, APOBEC3B also differs from APOBEC3F and APOBEC3G in that it is not normally expressed in the lymphoid cells that serve as targets for HIV-1 infection. These studies therefore raise the possibility that activation of the endogenous APOBEC3B gene in primary human lymphoid cells could form a novel and effective strategy for inhibition of HIV-1 replication in vivo.