Comparative Genomics: Mechanism(s) Against Emerging Infectious Diseases

比较基因组学：对抗新发传染病的机制

• 批准号: 7733073
• 负责人: STEPHEN J O'BRIEN
• 金额: $ 129.75万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733073
• 关键词: 5' Splice Site; Acquired Immunodeficiency Syndrome; African; Alternative Splicing; Amino Acid Substitution; Amino Acids; Anti-Retroviral Agents; Antigen-Presenting Cells; Antigens; Avian Influenza; Avian Influenza A Virus; Bacterial Artificial Chromosomes; Binding; Binding Sites; Blood; Breeding; CD4 Positive T Lymphocytes; Canis familiaris; Catalytic Domain; Cell Communication; Cell Line; Cells; Cercopithecus pygerythrus; Chiroptera; Chromosomal Breaks; Chromosome Arm; Chromosome inversion; Chromosomes; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 7; Class; Cloning; Code; DNA; DNA Sequence; Defense Mechanisms; Disease; Ebola Hemorrhagic Fever; Emerging Communicable Diseases; Endogenous Retroviruses; Evolution; Family Felidae; Feline Immunodeficiency Virus; Feline Leukemia Virus; Feline Syncytium-Forming Virus; Felis catus; Fibroblasts; Future; Gene Family; Genes; Genome; Genomics; HIV; HIV-1; HLA Antigens; Head; Helper-Inducer T-Lymphocyte; Hepatitis D; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Homologous Gene; Human; Hybrids; Immune system; Individual; Influenza; Influenza A Virus, H5N1 Subtype; Joining Exons; MHC Class I Genes; Major Histocompatibility Complex; Mammals; Marsupialia; Methods; Mutation; Nature; Numbers; Online Systems; Pan Genus; Pan troglodytes; Panthera leo; Papio; Peptides; Play; Polymerase Chain Reaction; Positioning Attribute; Proteins; Puma; Rate; Reading; Receptor Gene; Reporting; Retroviridae; Role; SLC26A3 gene; Sampling; Screening procedure; Serum; Severe Acute Respiratory Syndrome; Shotgun Sequencing; Shotguns; Single Nucleotide Polymorphism; Site; Site-Directed Mutagenesis; Skin; Spain; Structure; System; T-Lymphocyte; TRIM Gene; Tail; Terminator Codon; Time; Tissue Banks; Transient Global Amnesia; Translations; Viral Genome; Viral Physiology; Virus; Virus Diseases; Western Blotting; antigen processing; apolipoprotein B mRNA editing enzyme; base; comparative; cytotoxic; dimer; feline leukocyte antigen; genome sequencing; genome wide association study; immunological synapse; influenzavirus; olfactory receptor; pandemic disease; pathogen; prevent; programs; scaffold; tapasin; transmission process; vif Gene Products; virus host interaction; yeast two hybrid system

The major histocompatibility complex (MHC) plays key roles in controlling both adaptive and innate immune systems. In the adaptive immune system, both MHC class I and class II antigens recognize, bind and present peptides to cytotoxic and helper T-cells, respectively, and initiate cell-to-cell communication between antigen presenting cells and T-cells by forming immunological synapses and activating both subtypes of T-cells for cellular and humoral immune systems. More recently, a variety of host restriction genes have been identified in humans and mammals that modulate retrovirus infectivity, replication, assembly and/or cross-species transmission. One of these host encoded genes, Apolipoprotein B mRNA-editing enzyme catalytic (APOBEC2) is capable of terminally editing feline foamy virus in the absence of virally-encoded Bet protein, but not in its presence, similar to the interplay of APOBEC3 and the HIV-encoded protein Vif. The editing capacity of APOBEC3 appears to be species specific and limits cross-species transmission of retroviruses. To identify and characterize APOBEC genes in the feline genome, we attempted APOBEC-related sequences in the scaffolds of the partial (2x) genome sequence of the domestic cat and compared these phylogenetically to their human and dog counterparts. In addition, we determined approximately 50 kbp APOBEC3 region from three fosmid clones. (A) Comparative Genomic Structure of the MHC Comparisons of the genomic structure of three mammalian MHC, human leukocyte antigens (HLA), canine dog leukocyte antigens (DLA), and feline leukocyte antigens (FLA) revealed remarkable structural differences between HLA and the other two MHC. The 4.6 Mb HLA sequence was compared with the 3.9 Mb DLA sequence from two supercontigs generated by 7x whole genome shotgun assembly and 3.3 Mb FLA draft sequence. For FLA, we confirm that: (i) feline FLA was split into two pieces within the TRIM gene family found in human HLA; (ii) class I, II, and III regions were placed in the pericentrocentric region of the long arm of chromosome B2; and (iii) remaining FLA was located in subtelomeric region of the short arm of chromosome B2. The exact same chromosome break was found in canine DLA structure, where class I, II, and III regions were placed in a percentromeric region of chromosome 12, while the remaining region was located in a subtelomeric region of chromosome 35, suggesting this chromosome break occurred once before a split of felid and canid more than 55 MYA. However, significant differences were found in the content of genes in both pericentromeric and subtelomeric regions in DLA and FLA, the gene number and amplicon structure of class I genes plus two other class I genes found on two additional chromosomes; canine chromosome 7 and 18, suggests the dynamic nature in the evolution of MHC class I genes. (B) Sequences, Annotation and Single Nucleotide Polymorphism (SNP) of the MHC in the Domestic Cat Two sequences of the MHC regions in the domestic cat, 2.976 and 0.362 Mbps, which were separated by an ancient chromosome break (55 - 80 MYA) and followed by a chromosomal inversion were determined by bacterial artificial chromosome (BAC) shotgun sequencing. Gene annotation of this MHC was completed and identified 317 possible coding regions (128 human homologues, possible functional genes and 189 pseudo/unidentified genes) by GENSCAN, BLASTN, and BLASTP programs. The first region spans 2.976 Mbp sequence, which encodes six classical class II antigens (three DRA and three DRB antigens), nine antigen processing molecules (DOA/DOB, DMA/DMB, TAPASIN, and LMP2/LMP7.TAP1/TAP2), 52 class III genes, 19 class I antigens (FLAI-A to FLAI-S). Two class I genes (FLAI-H, I-K) were transcribed in a feline fibroblast cell line and one (FLAI-E) had a peptide binding site structure similar to the classical class I gene. The second region spans 0.362 Mbp sequence encoding no class I genes and 18 framework genes, including three olfactory receptor genes. One previously identified feline endogenous retrovirus, a baboon retrovirus derived sequence (ECE1) and two new endogeneous retrovirus sequences, both of which showed high sequence similarity to brown bat endogeneous retrovirus (FERVmlu1, FERVmlu2) were found within a 100 Kbp interval in the middle of class I region. MHC SNPs were examined based on comparisons of this BAC sequence and MHC homozygous 2 X whole genome scan (WGS) sequences and found that 11,654 SNPs in 2.84 Mbp (0.00411 SNP per bp), which is 2.4 times higher rate than average heterozygous region in 2 X WGS (0.0017 SNP per bp genome), and slightly higher than the SNP rate observed in human MHC (0.00337 SNP per bp). (C) Innate Defense Mechanisms Against Exogenous and Endogenous Retroviruses in the Domestic Cat. APOBEC3 anti retroviral function against three feline viruses (FIV, FeLV, Formy Virus). In order to find functions of APOBEC3 molecules, we have isolated three fosmid clones which cover approximately 50 kpb sequence on feline A3 locus using web-based fosmid cloning system established in feline genome browser (GARField). High quality and complete DNA sequences of these three fosmid clones were determined by transposon insertion and Sanger methods using Biomek Fx/ ABI3730XL DNA sequencer and Phred/Phrap/Consed applications. We found that a. Four A3C genes complete genes exist in head-tail fashions. b. Each A3Ca, A3Cb, A3c, A3H genes are transcribed and produce A3C molecules. c. Each molecules has differential antiretroviral activities. For example, A3a, A3b, A3c strongly suppress delta-bet formy virus infection, however no effects on infections of delta-vif FIV nor FeLV A3H suppress delta-vif FIV moderately but no effects on delta-bet formy virus nor FeLV. d. Read-through alternative splicing generate hybrid two catalytic domain A3CH molecules using cryptic splicing donor site overlapped with TGA stop codon of A3Cc and join exon 2 of A3H with in frame translation of A3H part of this hybrid molecule. This A3CH molecule have strong anti-viral activities against both delta-vif FIV and FeLV but not against delta-bet formy virus. e. Excess of nonsynonymous substitutions in A3C genes were found by SNP analyses of nine cat breeds. These amino acid positions are located on sites which involved in tetramer and dimer formation. One of these sites are found to be the same site which determine host specific interactions between human and african green monkey APOBEC3G and Vif in HIV and SIVagm. Site direct mutagenesis are now planned to examine the effects of these amino acid substitutions against FIV, FeLV and formy virus infections.

主要组织相容性复合体（MHC）在控制适应性和先天免疫系统中起着关键作用。在适应性免疫系统中，MHC I类和II类抗原分别识别、结合和呈递肽到细胞毒性和辅助性t细胞，并通过形成免疫突触和激活细胞和体液免疫系统的两种t细胞亚型来启动抗原呈递细胞和t细胞之间的细胞间通信。最近，在人类和哺乳动物中发现了多种宿主限制性基因，它们调节逆转录病毒的传染性、复制、组装和/或跨物种传播。其中一种宿主编码基因载脂蛋白B mrna编辑酶催化（APOBEC2）能够在没有病毒编码的Bet蛋白的情况下对猫泡沫病毒进行最终编辑，但在其存在时却不能，类似于APOBEC3和hiv编码蛋白Vif的相互作用。APOBEC3的编辑能力似乎是物种特异性的，并限制了逆转录病毒的跨物种传播。为了鉴定和表征猫基因组中的APOBEC基因，我们在家猫部分（2x）基因组序列的支架中尝试了apobecc相关序列，并将这些序列与人类和狗的对应序列进行了系统发育比较。此外，我们从三个fosmid克隆中确定了约50 kbp的APOBEC3区域。(A) MHC的基因组结构比较：对哺乳动物MHC、人白细胞抗原（HLA）、犬犬白细胞抗原（DLA）和猫白细胞抗原（FLA）的基因组结构进行比较，发现HLA与其他两种MHC在结构上存在显著差异。将4.6 Mb HLA序列与7次全基因组散弹枪组装生成的3.9 Mb DLA序列和3.3 Mb FLA草图序列进行比较。对于FLA，我们证实：(i)猫FLA在人类HLA中发现的TRIM基因家族中被分成两个片段；（ii） I、ii、III类区位于B2染色体长臂的近着心区；（iii）剩余的FLA位于染色体B2短臂的亚端粒区。在犬类的DLA结构中发现了完全相同的染色体断裂，其中I、II和III类区域位于12号染色体的百分分体区域，而其余区域位于35号染色体的亚端粒区域，这表明这种染色体断裂发生在55 MYA以上的猫科动物和犬科动物分裂之前。然而，DLA和FLA在近中心粒区和亚端粒区基因的含量、I类基因的数量和扩增子结构以及另外两条染色体上发现的另外两个I类基因的扩增子结构存在显著差异；犬的第7和18号染色体，提示MHC I类基因进化的动态性质。(B)家猫MHC序列、注释和单核苷酸多态性（SNP）采用细菌人工染色体（BAC）散弹枪测序法测定了家猫MHC区域的两个序列，分别为2.976和0.362 Mbps，这两个序列通过古染色体断裂（55 - 80 MYA）分离，然后进行染色体倒置。通过GENSCAN、BLASTN和BLASTP程序完成MHC的基因注释，鉴定出317个可能的编码区（128个人类同源基因、可能的功能基因和189个伪/未识别基因）。第一个区域全长2.976 Mbp，编码6个经典II类抗原（3个DRA和3个DRB抗原），9个抗原加工分子（DOA/DOB、DMA/DMB、TAPASIN和LMP2/LMP7）。TAP1/TAP2)， 52个III类基因，19个I类抗原（FLAI-A ~ FLAI-S）。两个I类基因（FLAI-H, I- k）在猫成纤维细胞系中转录，其中一个（FLAI-E）具有与经典I类基因相似的肽结合位点结构。第二区全长0.362 Mbp，无I类基因编码，18个框架基因编码，包括3个嗅觉受体基因。在I类区中部100 Kbp的间隔内发现了1个先前鉴定的猫内源性逆转录病毒、1个狒狒内源性逆转录病毒衍生序列（ECE1）和2个新的内源性逆转录病毒序列，均与棕蝠内源性逆转录病毒（FERVmlu1、FERVmlu2）具有高度的序列相似性。将该BAC序列与MHC纯合子2 X全基因组扫描（WGS）序列进行比较，发现2.84 Mbp中存在11,654个SNP (0.00411 SNP / bp)，是2 X WGS中平均杂合子区域（0.0017 SNP / bp）的2.4倍，略高于人类MHC的SNP （0.00337 SNP / bp）。(C)家猫对外源性和内源性逆转录病毒的先天防御机制。APOBEC3对三种猫病毒（FIV, FeLV, Formy Virus）的抗逆转录病毒功能。为了寻找APOBEC3分子的功能，我们利用猫基因组浏览器（GARField）中建立的基于web的fosmid克隆系统，分离了3个覆盖猫A3位点约50 kpb序列的fosmid克隆。使用Biomek Fx/ ABI3730XL DNA测序仪和Phred/Phrap/Consed应用程序，通过转座子插入和Sanger方法确定了这三个fosmid克隆的高质量和完整的DNA序列。我们发现a. 4个A3C基因完整基因以正反模式存在。b.每个A3Ca、A3Cb、A3c、A3H基因转录产生A3c分子。每种分子具有不同的抗逆转录病毒活性。例如，A3a、A3b、A3c对delta-bet formy病毒感染有较强的抑制作用，但对delta-vif FIV和FeLV感染没有作用。A3H对delta-vif FIV有中度抑制作用，但对delta-bet formy病毒和FeLV没有作用。d.通过选择性读接，利用与A3Cc的TGA终止密码子重叠的隐剪接供体产生杂交的两个催化结构域A3CH分子，并将A3H的外显子2与该杂交分子的A3H部分的框内翻译连接起来。该A3CH分子对δ -vif型FIV和FeLV均有较强的抗病毒活性，但对δ -bet型病毒没有较强的抗病毒活性。e.通过对9个猫品种的SNP分析，发现A3C基因中存在过量的非同义替换。这些氨基酸位置位于参与四聚体和二聚体形成的位点上。其中一个位点被发现与人类和非洲绿猴APOBEC3G和Vif在HIV和SIVagm中宿主特异性相互作用的位点相同。位点直接诱变现在计划检验这些氨基酸取代对FIV， FeLV和形虫病毒感染的影响。

项目成果

Feline immunodeficiency virus (FIV) in wild Pallas' cats.

• DOI: 10.1016/j.vetimm.2009.10.014
• 发表时间: 2010-03-15
• 期刊: Veterinary immunology and immunopathology
• 影响因子: 1.8
• 作者: Brown MA;Munkhtsog B;Troyer JL;Ross S;Sellers R;Fine AE;Swanson WF;Roelke ME;O'Brien SJ
• 通讯作者: O'Brien SJ