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发挥着关键作用。在适应性免疫系统中，MHC I 类和 II 类抗原分别识别、结合并向细胞毒性 T 细胞和辅助 T 细胞呈递肽，并通过形成免疫突触启动抗原呈递细胞和 T 细胞之间的细胞间通讯激活细胞和体液免疫系统的两种 T 细胞亚型。最近，在人类和哺乳动物中鉴定了多种宿主限制基因，它们调节逆转录病毒感染性、复制、组装和/或跨物种传播。这些宿主编码基因之一，载脂蛋白 B mRNA 编辑酶催化 (APOBEC2) 能够在病毒编码的 Bet 蛋白不存在的情况下最终编辑猫泡沫病毒，但在其存在的情况下则不能，类似于 APOBEC3 和 HIV 的相互作用-编码蛋白质Vif。 APOBEC3 的编辑能力似乎具有物种特异性，并限制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，我们尝试在家猫的部分 (2x) 基因组序列的支架中查找 APOBEC 相关序列，并将这些序列与人类和狗的对应序列进行系统发育比较。此外，我们从三个 fosmid 克隆中确定了大约 50 kbp APOBEC3 区域。 (A) MHC 的基因组结构比较 三种哺乳动物 MHC、人类白细胞抗原 (HLA)、犬白细胞抗原 (DLA) 和猫白细胞抗原 (FLA) 的基因组结构比较，发现 HLA 与其他 MHC 之间存在显着的结构差异。两个 MHC。将 4.6 Mb HLA 序列与来自 7x 全基因组鸟枪法组装生成的两个超级重叠群的 3.9 Mb DLA 序列和 3.3 Mb FLA 草图序列进行比较。对于 FLA，我们确认：(i) 猫科动物 FLA 在人类 HLA 中发现的 TRIM 基因家族中被分成两部分； (ii) I、II和III类区域位于B2染色体长臂的近中心区域； (iii)剩余的FLA位于B2染色体短臂的亚端粒区域。在犬科动物 DLA 结构中也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 号染色体的百分比区域，而其余区域位于 35 号染色体的亚端粒区域，表明发生了这种染色体断裂猫科动物和犬科动物分裂前一次超过 55 MYA。然而，DLA和FLA中着丝粒周围和亚端粒区域的基因含量、I类基因的基因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类基因的基因数量和扩增子结构存在显着差异；犬 7 号和 18 号染色体表明 MHC I 类基因进化的动态性质。 (B) 家猫 MHC 的序列、注释和单核苷酸多态性 (SNP) 家猫 MHC 区域的两个序列，2.976 和 0.362 Mbps，由古老的染色体断裂 (55 - 80 MYA) 分开然后通过细菌人工染色体（BAC）鸟枪测序确定染色体倒位。该MHC的基因注释已完成，并通过GENSCAN、BLASTN和BLASTP程序鉴定了317个可能的编码区（128个人类同源物、可能的功能基因和189个假/未鉴定基因）。第一个区域跨越 2.976 Mbp 序列，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 III 类基因，19 种 I 类抗原（FLAI-A 至 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。第二个区域跨越 0.362 Mbp 序列，不编码 I 类基因和 18 个框架基因，包括 3 个嗅觉受体基因。一种先前鉴定的猫科内源性逆转录病毒、一种狒狒逆转录病毒衍生序列（ECE1）和两种新的内源性逆转录病毒序列，两者均与棕蝙蝠内源性逆转录病毒（FERVmlu1、FERVmlu2）表现出高度的序列相似性，在2017年中期的100 Kbp间隔内发现。 I类区域。基于该 BAC 序列和 MHC 纯合 2 X 全基因组扫描 (WGS) 序列的比较，检查了 MHC SNP，发现 2.84 Mbp 中有 11,654 个 SNP（每 bp 0.00411 个 SNP），比 2 中平均杂合区域高出 2.4 倍。 X WGS（每个 bp 基因组 0.0017 个 SNP），略高于在人类 MHC（每个 bp 0.00337 个 SNP）。 (C) 家猫针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 病毒）具有抗逆转录病毒功能。为了寻找APOBEC3分子的功能，我们使用在猫科动物基因组浏览器（GARField）中建立的基于网络的fosmid克隆系统分离了三个覆盖猫A3基因座上约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 DNA 序列。我们发现，a.四个A3C基因完整基因以头尾方式存在。 b.每个A3Ca、A3Cb、A3c、A3H基因都被转录并产生A3C分子。 c.每个分子都具有不同的抗逆转录病毒活性。例如，A3a、A3b、A3c强烈抑制delta-bet福米病毒感染，但对delta-vif FIV或FeLV感染没有影响。 A3H中度抑制delta-vif FIV，但对delta-bet福米病毒或FeLV没有影响。 d.通读选择性剪接使用与 A3Cc 的 TGA 终止密码子重叠的隐秘剪接供体位点生成杂合两个催化结构域 A3CH 分子，并将 A3H 的外显子 2 与该杂合分子的 A3H 部分的框内翻译连接起来。这种 A3CH 分子对 delta-vif FIV 和 FeLV 均具有很强的抗病毒活性，但对 delta-bet formy 病毒没有作用。 e.通过对 9 个猫品种的 SNP 分析发现 A3C 基因中存在过多的非同义替换。这些氨基酸位置位于参与四聚体和二聚体形成的位点。发现这些位点之一与决定人类和非洲绿猴 APOBEC3G 和 HIV 和 SIVagm 中 Vif 之间宿主特异性相互作用的位点相同。现在计划通过定点诱变来检查这些氨基酸取代对 FIV、FeLV 和甲型病毒感染的影响。

项目成果

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

野生帕拉斯猫中的猫免疫缺陷病毒（FIV）。

• 发表时间: 2010-03-15
• 影响因子: 1.8
• 作者: Brown, Meredith A;Munkhtsog, Bariushaa;Troyer, Jennifer L;Ross, Steve;Sellers, Rani;Fine, Amanda E;Swanson, William F;Roelke, Melody E;O'Brien, Stephen J
• 通讯作者: O'Brien, Stephen J