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: 7592749
• 负责人: STEPHEN J O'BRIEN
• 金额: $ 48.48万
• 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/7592749
• 关键词: Acquired Immunodeficiency Syndrome; African; Alternative Splicing; Antigen-Presenting Cells; Antigens; Avian Influenza; Avian Influenza A Virus; Bacterial Artificial Chromosomes; Binding; Binding Sites; Blood; CD4 Positive T Lymphocytes; Canis familiaris; Cell Communication; Cell Line; Cells; Chiroptera; Chromosomal Breaks; Chromosome Arm; Chromosome inversion; Chromosomes; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 7; Class; Code; DNA; Disease; Ebola Hemorrhagic Fever; Emerging Communicable Diseases; Endogenous Retroviruses; Evolution; Family Felidae; Feline Immunodeficiency Virus; Feline Syncytium-Forming Virus; Felis catus; Fibroblasts; Future; Gene Duplication; Gene Family; Genes; Genome; Genomics; HIV; HIV-1; HLA Antigens; Helper-Inducer T-Lymphocyte; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Homologous Gene; Human; Immune system; Individual; Influenza; Influenza A Virus, H5N1 Subtype; MHC Class I Genes; Major Histocompatibility Complex; Mammals; Marsupialia; Messenger RNA; Methods; Mutation; Nature; Numbers; Pan Genus; Pan troglodytes; Panthera leo; Papio; Peptides; Phylogeny; Play; Polymerase Chain Reaction; Primates; Proteins; Puma; Rate; Reading; Receptor Gene; Reporter; Reporting; Retrotransposon; Retroviridae; Rodent; Role; SLC26A3 gene; Sampling; Screening procedure; Serum; Severe Acute Respiratory Syndrome; Shotgun Sequencing; Shotguns; Single Nucleotide Polymorphism; Site; Skin; Spain; Structure; Subgroup; System; T-Lymphocyte; TRIM Gene; Time; Tissue Banks; Viral Genome; Virus; Western Blotting; antigen processing; apolipoprotein B mRNA editing enzyme; base; comparative; cytotoxic; feline leukocyte antigen; genome sequencing; genome wide association study; immunological synapse; influenzavirus; inhibitor/antagonist; leukemia virus; olfactory receptor; pandemic disease; pathogen; polypeptide; prevent; programs; scaffold; tapasin; transmission process; vif Gene Products; virus host interaction

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. (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) Functions, Structure, and Read-Through Alternative Splicing of Feline APOBEC3 Genes Over the last few years, a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly and/or cross-species transmission. One of the host encoded proteins, APOBEC3 (A3, Apoliporoteins B mRNA-editing catalytic polypeptide) is a potent inhibitor of retroviruses and retrotransposons. While primates encode seven genes (A3A to H), rodents carry only a single A3 gene. Here we identified and characterized several A3 genes in the feline genome by characterizing the 50k genomic A3 locus. We detected that the domestic cat (<i>Felis catus</i>) has three very similar A3C genes (a c) probably generated by two consecutive gene duplications and one A3H gene. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by a read-through alternative splicing. Using reporter systems for feline retroviruses, we found that specific feline A3 proteins selectively inactivate only defined subgroups of feline retroviruses: Bet-deficient feline foamy virus was inactivated by feA3Ca, -b and c, but not by feA3H or feA3CH. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. We compared the phylogeny of cat A3s with their counterparts in other felid species. As anticipated, the feline A3C sequences show significant adaptive evolution, but unexpectedly, the A3H sequences contain more sites which are under purifying evolution probably reflecting differences in the respective host virus co-evolution. In contrast to cats, dogs encode only A3A and A3H which may be related to the fact that there are no exogenous retroviruses of dogs present

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并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讯。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 编码蛋白 Vif 的相互作用。 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。 (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) 的基因组结构的比较揭示了 HLA 与其他两种 MHC 之间显着的结构差异。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 号染色体的百分体区域，而其余区域位于 35 号染色体的亚端粒区域，表明这种染色体断裂在猫科动物和犬科动物分裂超过 55 MYA 之前发生过一次。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，通过细菌人工染色体 (BAC) 鸟枪测序确定，这两个序列通过古老染色体断裂 (55 - 80 MYA) 分开，然后进行染色体倒位。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种经典II类抗原（3种DRA和3种DRB抗原）、9种抗原加工分子（DOA/DOB、DMA/DMB、TAPASIN和LMP2/LMP7.TAP1/TAP2）、52种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。在 I 类区域中部 100 Kbp 的间隔内发现了一种先前鉴定的猫内源性逆转录病毒、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，这两种序列均与棕蝙蝠内源性逆转录病毒 (FERVmlu1、FERVmlu2) 具有高度的序列相似性。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 SNP），这是 2 X WGS 中平均杂合区域（每 bp 基因组 0.0017 SNP）的 2.4 倍，略高于在 2 X WGS 中观察到的 SNP 率。人类的 MHC（每个碱基对 0.00337 个 SNP）。 (C) 猫 APOBEC3 基因的功能、结构和通读选择性剪接 在过去的几年中，已在人类和哺乳动物中鉴定出多种调节逆转录病毒感染性、复制、组装和/或跨物种传播的宿主限制基因。 APOBEC3（A3，载脂蛋白 B mRNA 编辑催化多肽）是宿主编码的蛋白质之一，是逆转录病毒和逆转录转座子的有效抑制剂。灵长类动物编码七个基因（A3A 到 H），而啮齿动物只携带一个 A3 基因。在这里，我们通过表征 50k 基因组 A3 位点，鉴定并表征了猫基因组中的几个 A3 基因。我们检测到家猫 (<i>Felis catus</i>) 具有 3 个非常相似的 A3C 基因 (a c)，可能是由两个连续的基因重复产生的，以及一个 A3H 基因。除了这四种单结构域 A3 蛋白之外，第五种 A3（称为 A3CH）通过通读选择性剪接表达。使用猫科逆转录病毒报告系统，我们发现特定的猫科 A3 蛋白仅选择性地灭活特定的猫科逆转录病毒亚群：Bet 缺陷猫泡沫病毒可被 feA3Ca、-b 和 c 灭活，但不会被 feA3H 或 feA3CH 灭活。 Vif缺陷的猫免疫缺陷病毒和猫白血病病毒的感染性仅被feA3H和feA3CH降低，但不被任何feA3C降低。我们将猫 A3 的系统发育与其他猫科动物的对应物种进行了比较。正如预期的那样，猫科动物的 A3C 序列显示出显着的适应性进化，但出乎意料的是，A3H 序列包含更多处于纯化进化中的位点，这可能反映了各自宿主病毒共同进化的差异。与猫不同，狗只编码A3A和A3H，这可能与狗不存在外源性逆转录病毒有关。