Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.     

Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded beta-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.     

Retroviral invasion of the koala genome

Endogenous retroviruses are a common ancestral feature of mammalian genomes with most having been inactivated over time through mutation and deletion. A group of more intact endogenous retroviruses are considered to have entered the genomes of some species more recently, through infection by exogenous viruses, but this event has never been directly proved. We have previously reported koala retrovirus (KoRV) to be a functional virus that is associated with neoplasia. Here we show that KoRV also shows features of a recently inserted endogenous retrovirus that is vertically transmitted. The finding that some isolated koala populations have not yet incorporated KoRV into their genomes, combined with its high level of activity and variability in individual koalas, suggests that KoRV is a virus in transition between an exogenous and endogenous element. This ongoing dynamic interaction with a wild species provides an exciting opportunity to study the process and consequences of retroviral endogenization in action, and is an attractive model for studying the evolutionary event in which a retrovirus invades a mammalian genome.     

APOBEC3 inhibits mouse mammary tumour virus replication in vivo

Genomes of all mammals encode apobec3 genes, which are thought to have a function in intrinsic cellular immunity to several viruses including human immunodeficiency virus type 1 (HIV-1). APOBEC3 (A3) proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytidines on the negative strand DNA intermediates. However, the role of A3 in innate resistance to mouse retroviruses is not understood. Here we show that A3 functions during retroviral infection in vivo and provides partial protection to mice against infection with mouse mammary tumour virus (MMTV). Both mouse A3 and human A3G proteins interacted with the MMTV nucleocapsid in an RNA-dependent fashion and were packaged into virions. In addition, mouse A3-containing and human A3G-containing virions showed a marked decrease in titre. Last, A3(-/-) mice were more susceptible to MMTV infection, because virus spread was more rapid and extensive than in their wild-type littermates.     

The APOBEC-2 crystal structure and functional implications for the deaminase AID

APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC protein called activation-induced cytidine deaminase (AID) is critical for generating high-affinity antibodies, and deamination by APOBEC-3 proteins can inhibit retrotransposons and the replication of retroviruses such as human immunodeficiency virus and hepatitis B virus. Here we report the crystal structure of APO2. APO2 forms a rod-shaped tetramer that differs markedly from the square-shaped tetramer of the free nucleotide cytidine deaminase, with which APOBEC proteins share considerable sequence homology. In APO2, two long alpha-helices of a monomer structure prevent the formation of a square-shaped tetramer and facilitate formation of the rod-shaped tetramer via head-to-head interactions of two APO2 dimers. Extensive sequence homology among APOBEC family members allows us to test APO2 structure-based predictions using AID. We show that AID deamination activity is impaired by mutations predicted to interfere with oligomerization and substrate access. The structure suggests how mutations in patients with hyper-IgM-2 syndrome inactivate AID, resulting in defective antibody maturation.     

胞嘧啶脱氨基酶APOBEC1研究进展

为全面理解载脂蛋白B mRNA(ApoB mRNA)编辑酶催化多肽-1(APOBEC1)的作用机制,介绍了APOBEC1和ApoB mRNA的蛋白及核酸序列,总结并绘制了APOBEC1与不同的辅助蛋白的结合模型,阐述了APOBEC1催化ApoB mRNA第6 666位的胞嘧啶(C_(6666))脱氨基化分子机制.列举了啮齿动物APOBEC1抑制多种逆转录病毒的研究报道,介绍了兔源APOBEC1结合人类免疫缺陷病毒1(HIV-1)的病毒粒子并编辑病毒基因组的机理.同时介绍了APOBEC1通过编辑胞嘧啶或与AU富集元件(ARE)结合来调控癌症等疾病相关的细胞因子表达.     

Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution

Interactions between bacterial hosts and their viruses (phages) lead to reciprocal genome evolution through a dynamic co-evolutionary process. Phage-mediated transfer of host genes--often located in genome islands--has had a major impact on microbial evolution. Furthermore, phage genomes have clearly been shaped by the acquisition of genes from their hosts. Here we investigate whole-genome expression of a host and phage, the marine cyanobacterium Prochlorococcus MED4 and the T7-like cyanophage P-SSP7, during lytic infection, to gain insight into these co-evolutionary processes. Although most of the phage genome was linearly transcribed over the course of infection, four phage-encoded bacterial metabolism genes formed part of the same expression cluster, even though they are physically separated on the genome. These genes--encoding photosystem II D1 (psbA), high-light inducible protein (hli), transaldolase (talC) and ribonucleotide reductase (nrd)--are transcribed together with phage DNA replication genes and seem to make up a functional unit involved in energy and deoxynucleotide production for phage replication in resource-poor oceans. Also unique to this system was the upregulation of numerous genes in the host during infection. These may be host stress response genes and/or genes induced by the phage. Many of these host genes are located in genome islands and have homologues in cyanophage genomes. We hypothesize that phage have evolved to use upregulated host genes, leading to their stable incorporation into phage genomes and their subsequent transfer back to hosts in genome islands. Thus activation of host genes during infection may be directing the co-evolution of gene content in both host and phage genomes.     

Interferon inhibits transformation by murine sarcoma viruses before integration of provirus

Neoplastic transformation by C-type retroviruses requires synthesis of a DNA copy (the provirus) of the RNA genome and its integration into the host cell DNA. We have previously shown that interferon (IFN) can stably prevent transformation of murine fibroblasts by the Kirsten strain of murine sarcoma virus (KiMSV), a murine leukaemia virus (MLV). A series of cell clones (IFN clones), isolated in the presence of IFN (10(4) U ml-1) from cultures of NIH-3T3 cells which had been treated with IFN, and then infected with KiMSV (KiMLV) in conditions where every cell was infected, were shown to be phenotypically untransformed. These untransformed cells did not produce virus or contain rescuable KiMSV. However, cells isolated using an identical procedure, but in the absence of IFN, were uniformly transformed and all produced KiMSV (KiMLV) or contained rescuable KiMSV. It was concluded that IFN either prevents synthesis or integration of the provirus, or else that in the presence of IFN the provirus is integrated such that it is not expressed. We now show that five representative clones contain no detectable KiMSV proviral DNA, and also that the initial stages of infection by KiMSV (KiMLV) are inhibited by IFN treatment. IFN seems to act before integration, preventing either the synthesis or the integration of proviral DNA.     

Severe immunodeficiency disease induced by a defective murine leukaemia virus

Different classes of retroviruses have been shown to induce immunodeficiency diseases in various animal species. These animal models may provide an insight into our understanding of AIDS but, with the exception of one strain of feline leukaemia virus, the determinants of pathogenicity have not yet been mapped to these viral genomes. The immunodeficiency-inducing feline leukaemia virus is replication-defective, harbouring the determinant of pathogenicity within its env sequences. We have studied the Duplan strain of murine leukaemia virus which induces, in C57BL/6 mice, a severe immunodeficiency disease with striking similarities to human AIDS. We have identified the aetiological agent of this murine immunodeficiency disease as another defective retrovirus, with a genome of 4.8 kilobases. Molecular cloning and sequencing of this DNA showed that the pol and env genes have been deleted, but that the complete gag region has been conserved and has a novel sequence encoding the p12 protein. As with the feline leukaemia virus, these results provide evidence for the role of defective retroviruses in inducing immunodeficiency and facilitate the study of the mechanisms underlying the pathogenesis of retrovirus-induced immunodeficiency syndromes, including AIDS.     

Identification of novel SINEs from Cyprinidae and their evolutionary significance

Short interspersed nuclear elements (SINEs) are widespread among eukaryotic genomes. They are repetitive DNA sequences that have been amplified by retrotransposition. In this study, a class of SINEs were isolated from the Opsariichthys bidens genome, and named Opsar. Sequence analysis confirmed that Opsar is a new class of typical SINEs derived from tRNA molecules. With the tRNA-derived region of Opsar and through BLASTN search, we further identified Zb-SINEs from the zebrafish genome, which includes two groups: Zb-SINE-A and Zb-SINE-B. The Zb-SINE-A group comprises subfamilies of -A1—-A5, and the Zb-SINE-B group is a dimer of the tRNA^Ala-derived region and shares a similar dimeric composition to Alu. Zb-SINEs are composed of three distinct regions: a 5′end tRNA-derived region, a tRNA-unrelated region and a 3′end AT-rich region. The flanking regions are AT rich. The average length of Zb-SINEs elements is about 340 bp. Zb-SINEs account for as much as 0.1% of the whole zebrafish genome. About 70% of the Zb-SINEs are on chromosomes 11, 18, and 19. These Zb-SINEs were characterized by PCR and dot hybridization. The distribution pattern of Zb-SINEs in genome strongly supports the master genes model. The tRNA-derived regions of Opsar and Zb-SINEs were compared with the tRNA^Ala gene, and they showed 76% similarity, indicating that Opsar and Zb-SINEs originated from an inactive tRNA^Ala sequence or a tRNA^Ala—like sequence. In view of the evolutionary status of zebrafish in the Cyprinidae, we deduced that Zb-SINEs were a very old class of interspersed sequences.     

Identification of novel SINEs from Cyprinidae and their evolutionary significance

Short interspersed nuclear elements (SINEs) are widespread among eukaryotic genomes. They are repetitive DNA sequences that have been amplified by retrotransposition. In this study, a class of SINEs were isolated from the Opsariichthys bidens genome, and named Opsar. Sequence analysis confirmed that Opsar is a new class of typical SINEs derived from tRNA molecules. With the tRNA-derived region of Opsar and through BLASTN search, we further identified Zb-SINEs from the zebrafish genome, which includes two groups: Zb-SINE-A and Zb-SINE-B. The Zb-SINE-A. group comprises subfamilies of -Al--A5, and the Zb-SINE-B group is a dimer of the tRNA -derived region and shares a similar dimeric composition to Alu. Zb-SINEs are composed of three distinct regions: a 5' end tRNA-derived region, a tRNA-unrelated region and a 3' end AT-rich region. The flanking regions are AT rich. The average length of Zb-SINEs elements is about 340 bp. Zb-SINEs account for as much as 0.1 % of the whole zebrafish genome. About 70% of the Zb-SINEs are on chromosomes 11, 18, and 19. These Zb-SINEs were characterized by PCR and dot hybridization. The distribution pattern of Zb-SINEs in genome strongly supports the master genes model. The tRNA-derived regions of Opsar and Zb-SINEs were compared with the tRNAAla gene, and they showed 76% similarity, indicating that Opsar and Zb-SINEs originated from an inactive tRNA sequence or a tRNA -like sequence. In view of the evolutionary status of zebrafish in the Cyprinidae, we deduced that Zb-SINEs were a very old class of interspersed sequences.