High expression of APOBEC3G in patients infected with hepatitis C virus

APOBEC3G (an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a member of the APOBEC family, which possesses cytidine deaminase activity that causes C/G to T/A transition mutations in virus genomes such as human immunodeficiency virus 1 and hepatitis B virus, is reported to play an important role in host-defense mechanisms. However, APOBEC3G expression in patients infected with chronic hepatitis C virus (HCV), of which there are currently more than 170 million worldwide, has not yet been well studied. We investigated this issue herein, and demonstrated an increased expression of APOBEC3G in both hepatocytes and lymphocytes of chronic hepatitis patients infected with HCV. Transfection of the NS5A gene, but not any other non-structural protein genes of HCV tested, to the hepatocellular carcinoma cell line enhanced APOBEC3G expression. Incubation of the cells with interferon also resulted in the augmentation. These results may provide new insight into the pathogenesis of chronic HCV infection.     

艾滋病患者APOBEC3G 基因的克隆和真核表达

目的 建立艾滋病( AIDS) 患者载脂蛋白B mRNA 编辑酶催化多肽样蛋白3G( APOBEC3G) 的真核表达体系。方法 采用反转录-聚合酶链反应( RT-PCR) 技术从AIDS 患者外周血单个核细胞( PBMC) 中获取APOBEC3G 基因编码区, 将其克隆到pMD18-T载体上, 测序验证正确后再将其转接入真核表达载体pEGFP-N1 中, 然后将重组质粒pEGFP-N1-A3G 转染HEK293T细胞, 分别用RT-PCR 法和蛋白印迹法( Western 印迹法) 验证APOBEC3G 在mRNA 和蛋白水平的表达。结果 从AIDS 患者体内克隆的APOBEC3G 基因编码区长度为1 154 bp, 测序结果与GenBank 中APOBEC3G 参考序列( NM021822) 比对发现存在2 处差异, 分别位于mRNA 第588 位和746 位碱基处。重组质粒pEGFP-N1-A3G转染HEK293T 细胞, 在荧光显微镜下观察到融合蛋白A3G-EGFP的表达, RT-PCR 法和Western blot 法分别验证了蛋白在mRNA 和蛋白水平的表达。结论 成功构建了AIDS 患者APOBEC3G 蛋白的真核表达体系, 为进一步研究APOBEC3G 在HIV-1 感染中的作用奠定了基础。     

小鼠APOBEC3的结构与功能分析

随着对APOBEC3抗病毒功能的深入研究以及其在癌症中的表现,APOBEC3已成为当前的热点.小鼠只有一个APOBEC3基因,而人类有七个APOBEC3基因,人类APOBEC3G是其中研究最明确的抗病毒蛋白.利用生物信息学方法对两个蛋白进行序列比对、亲疏水性分析、亚细胞定位预测、二级结构及高级结构分析以及相互作用分析....     

Extensive mutagenesis experiments corroborate a structural model for the DNA deaminase domain of APOBEC3G

APOBEC3G is a single-strand DNA cytosine deaminase capable of blocking retrovirus and retrotransposon replication. APOBEC3G has two conserved zinc-coordinating motifs but only one is required for catalysis. Here, deletion analyses revealed that the minimal catalytic domain consists of residues 198-384. Size exclusion assays indicated that this protein is monomeric. Many (31/69) alanine substitution derivatives of APOBEC3G198-384 retained significant to full levels of activity. These data corroborated an APOBEC2-based structural model for the catalytic domain of APOBEC3G indicating that most non-essential residues are solvent accessible and most essential residues cluster within the protein core.     

Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short (> or =10 nucleotides) single-stranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.     

APOBEC3G蛋白的原核表达及纯化

目的：原核表达重组APOBEC3G蛋白,为其功能及免疫原性研究奠定基础。方法：提取H9细胞全细胞基因组RNA,通过RT-PCR获得目的基因,经纯化、酶切后克隆到原核表达载体pET32a中,转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,并对表达条件和纯化条件进行优化;利用Western Blot分析鉴定目的蛋白。结果：构建了APOBEC3G蛋白的原核表达载体Apo-His-pET32a,并在大肠杆菌中获得高表达,目的蛋白以可溶性蛋白形式存在;经Ni-NTA亲和层析柱一步纯化,获得了高纯度的重组APOBEC3G蛋白,蛋白浓度可达1．2mg／mL;Westem Blot显示获得了目的蛋白。结论：在原核表达系统中表达、纯化了可溶性APOBEC3G蛋白,为进一步对其进行免疫原性和功能研究奠定了基础。     

Human endogenous retroviruses, hormones and APOBEC3G: A connection to explore in schizophrenia

Human endogenous retroviruses (HERVs) represent the footprints of previous retroviral infections. They are integrated within the human germ line and constitute approximately 7% of our genome. They have the potential to harm, given their capacity to alter the cellular metabolism, and could be involved in various pathological processes such as systemic lupus erythematosus or multiple sclerosis. In this respect it has been found that the stimulation of HERVs genome expression was observed after a steroid hormone treatment, stating the first evidence that an enhanced expression of the HERVs genome by hormones may be involved in the etiology of breast cancer. There is now increasing evidence that HERVs may in fact be involved in the etiology of schizophrenia, a disorder characterized by heterogeneous presence of positive, negative and cognitive symptoms that affect all aspects of mental activity, with a first peak incidence for males and females in the decade 15-24 and a second peak at age 55-64 for females, both periods characterized by two moments of significant hormonal changes. In connection with genetic aspects, several studies suggest a linkage between chromosome 22 (22q) and schizophrenia, being different genes of this chromosomal region reported as candidate genes for association with the disorder. Likewise, in a closely region of these genes, on 22q13, is located a gene named APOBEC3G, a potent intrinsic inhibitor of retroviral replication that also includes some HERVs. We propose that hormonal changes that coincide with two peak incidences in schizophrenia produce an enhancement in the expression of some HERV families implicated in the etiopathology of the disorder. The expression of HERVs is followed by a defective action of APOBEC3G that avoids carry out its function, that is, the inhibition of retroviral replication. This altered process might play a critical role in the etiopathogenesis of schizophrenia.     

HIV-1 Vif protein mediates the degradation of APOBEC3G in fission yeast when over-expressed using codon optimization

Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S.pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.     

Characterization of conserved motifs in HIV-1 Vif required for APOBEC3G and APOBEC3F interaction

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, or A3G) and related cytidine deaminases such as apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F) are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus (HIV)-1 requires suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through a common mechanism by recruiting Cullin5, ElonginB, and ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. Domains in Vif that mediate APOBEC3 recognition have not been fully characterized. In the present study, we identified a VxIPLx_4-5LxΦx₂YWxL motif in HIV-1 Vif, which is required for efficient interaction between Vif and A3G, Vif-mediated A3G degradation and virion exclusion, and functional suppression of the A3G antiviral activity. Amino acids 52 to 72 of HIV-1 Vif (including the VxIPLx_4-5LxΦx₂YWxL motif) alone could mediate interaction with A3G, and this interaction was abolished by mutations of two hydrophobic amino acids in this region. We have also observed that a Vif mutant was ineffective against A3G, yet it retained the ability to interact with Cullin5-E3 ubiquitin complex and A3G, suggesting that interaction with A3G is necessary but not sufficient to inhibit its antiviral function. Unlike the previously identified motif of HIV-1 Vif amino acids 40 to 44, which is only important for A3G suppression, the VxIPLx_4-5LxΦx₂YWxL motif is also required for efficient A3F interaction and suppression. On the other hand, another motif, TGERxW, of HIV-1 Vif amino acids 74 to 79 was found to be mainly important for A3F interaction and inhibition. Both the VxIPLx_4-5LxΦx₂YWxL and TGERxW motifs are highly conserved among HIV-1, HIV-2, and various simian immunodeficiency virus Vif proteins. Our data suggest that primate lentiviral Vif molecules recognize their autologous APOBEC3 proteins through conserved structural features that represent attractive targets for the development of novel inhibitors.     

Expression of APOBEC2 is transcriptionally regulated by NF-kappaB in human hepatocytes

Apolipoprotein B mRNA-editing enzyme catalytic subunit 2 (APOBEC2) is a member of the nucleic-acid-editing enzymes. However, the physiological function of APOBEC2 remains unclear. We demonstrate that APOBEC2 expression is strongly enhanced in response to both tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta. Inhibition of NF-kappaB activation invariably blocks TNF-alpha-induced APOBEC2 expression. The promoter region of APOBEC2 contains functional NF-kappaB response elements in the 5' untranslated region of the gene at -625/-616. These results show that APOBEC2 expression is regulated by pro-inflammatory cytokines via NF-kappaB activation and suggest a possible role of APOBEC2 in the pathophysiology of hepatic inflammation.     

Selective inhibition of Alu retrotransposition by APOBEC3G

The non-LTR retrotransposon LINE-1 (L1) comprises  17% of the human genome, and the L1-encoded proteins can function in trans to mediate the retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other L1-mediated retrotransposition processes. We demonstrate that APOBEC3G selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An active cytidine deaminase site is not required for the inhibition of Alu retrotransposition and the resultant integration events lack G to A or C to T hypermutation. These data demonstrate a differential restriction of L1 and Alu retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein complex may be targeted by APOBEC3G.     

激光扫描共聚焦显微镜研究APOBEC3G蛋白的亚细胞定位

采用RT-PCR技术,从HIV的非允许性H9细胞中获得载脂蛋白B mRNA编辑酶催化多肽样蛋白3G(APOBEC3G)的全长cDNA。APOBEC3G cDNA全长1 155nt,编码384个氨基酸。将APOBEC3G克隆到真核表达载体pEGFP-C3上,转染CD4 HeLa细胞,激光扫描共聚焦显微镜下可观察到表达的GFP-APOBEC3G融合蛋白定位于细胞质。     

Apo2L/TRAIL is an indirect mediator of apoptosis induced by interferon-alpha in human myeloma cells

Interferon-alpha (IFN-alpha) is currently used for the therapy of multiple myeloma (MM) though it is only effective in some patients. IFN-alpha induces apoptosis in some MM cell lines and it has been proposed to occur through an autocrine loop involving Apo2L/TRAIL. We have analysed the sensitivity to IFN-alpha and Apo2L/TRAIL of five MM cell lines and found no correlation between the apoptosis inducing ability of both cytokines. IFN-alpha-induced apoptosis in MM cells was not prevented by a caspase-8 selective inhibitor (Z-IETD-fmk) or blocking Apo2L/TRAIL. However, human monocytes treated with IFN-alpha release bioactive Apo2L/TRAIL to culture media which was cytotoxic for MM cells resistant to IFN-alpha. We propose that Apo2L/TRAIL released from IFN-alpha-stimulated blood monocytes would be a major mediator of the anti-myeloma effect of IFN-alpha in vivo.