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: 7965401
• 负责人: STEPHEN J O'BRIEN
• 金额: $ 99.46万
• 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/7965401
• 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; 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; 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; Online Systems; Pan Genus; Panthera leo; Papio; Peptides; Play; Positioning Attribute; Proteins; Puma; Reading; Receptor Gene; Reporting; Retroviridae; Role; 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; 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.

主要的组织相容性复合物（MHC）在控制适应性和先天免疫系统方面起着关键作用。在自适应免疫系统中，MHC I类和II类抗原分别识别，结合并呈现肽与细胞毒性和辅助辅助性T细胞，并通过形成免疫突触并激活T-Cells的T-Cells t-Cells of T-Cells的细胞和T细胞之间的细胞与T细胞之间的细胞对细胞通信。最近，已经在调节逆转录病毒感染，复制，组装和/或跨物种传播的人类和哺乳动物中鉴定了各种宿主限制基因。这些宿主编码的基因之一，载脂蛋白B mRNA编辑酶催化（APOBEC2）能够在没有病毒编码的BET蛋白的情况下终止编辑猫泡沫病毒，但与Apobec3和Hiv-Apbobec3和HIV蛋白质的互动类似。 APOBEC3的编辑能力似乎是特定物种的，并且限制了逆转录病毒的跨物种传播。为了识别和表征猫科动物基因组中的Apobec基因，我们尝试了家族CAT的部分（2X）基因组序列的APOBEC相关序列，并将这些系统发育与人类和狗的对应物进行比较。此外，我们从三个fosmid克隆确定了大约50 kbp apobec3区域。 （a）三种哺乳动物MHC，人白细胞抗原（HLA），犬类狗白细胞抗原（DLA）和猫白细胞抗原（FLA）的MHC比较的比较基因组结构，揭示了HLA和HLA之间的显着结构差异。将4.6 MB HLA序列与由7倍全基因组shot弹枪装配产生的两个超脑的3.9 Mb DLA序列和3.3 MB FLA绘制序列进行了比较。对于FLA，我们确认：（i）在人类HLA中发现的修剪基因家族中，猫素FLA分为两块； （ii）将I，II和III级区域放置在B2染色体长臂的中心区域； （iii）剩余的FLA位于染色体B2短臂的亚电体区域。在犬DLA结构中发现了完全相同的染色体断裂，其中I类，II和III级区域被放置在12号染色体染色体的一个百分比区域，而其余区域则位于35染色体的下层中，这表明该染色体破裂一次是在羊毛和罐头分裂之前发生的一次。然而，在DLA和FLA中的周围粒细胞和亚te脑区域的基因含量中发现了显着差异。犬类染色体第7和18号表明，MHC I类基因的进化中的动态性质。 (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.该MHC的基因注释已完成，并确定了317个可能的编码区域（128个人类同源物，可能的功能基因和189个伪/未识别基因），由Genscan，BlastN和BLASTP程序。 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).在猫成纤维细胞系中转录了两个I类基因（Flai-H，I-K），一个（FlaI-E）具有类似于经典I类基因的肽结合位点结构。第二个区域跨越了0.362 MBP序列，编码没有I类基因和18个框架基因，包括三个嗅觉受体基因。一个先前鉴定出的猫内源性逆转录病毒，狒狒逆转录病毒衍生序列（ECE1）和两个新的内差逆转录病毒序列，在100 kbp Inderd Indimed I区域中，发现了与棕色蝙蝠逆转录病毒（FervMlu1，fervmlu2）相似的高序列。根据该BAC序列的比较和MHC纯合2 x全基因组扫描（WGS）序列检查MHC SNP，发现11,654个SNP在2.84 Mbp中（0.00411 snp）（每BP 0.00411 SNP），是2 x WG的平均杂质（0.001 snp and Band and anp and bande and bane）中的杂合率较高的速率（均为0.0017 snp），该速率均高于平均差异。 MHC（每BP 0.00337 SNP）。 （c）针对家猫的外源性和内源性逆转录病毒的先天防御机制。 APOBEC3抗逆转录病毒功能针对三种猫病毒（FIV，FELV，方向病毒）。为了找到APOBEC3分子的功能，我们使用了基于Web的基因组浏览器（Garfield）中建立的基于Web的Fosmid克隆系统，分离了三个fosmid克隆，覆盖了猫A3基因座上约50 kpb序列。这三个fosmid克隆的高质量和完整的DNA序列是通过转座插入和使用Biomek FX/ABI3730XL DNA Sequencer和PHRED/PHRAP/CASS COSS应用的应用确定的。我们发现一个。头尾时尚中存在四个A3C基因完整基因。 b。每个A3CA，A3CB，A3C，A3H基因都被转录并产生A3C分子。 c。每个分子具有鉴别抗逆转录病毒活性。例如，A3a，A3b，A3C强烈抑制了Delta-bet福型病毒感染，但是对Delta-VIF FIF FIF FIF和FELV A3H的感染没有影响，也不会抑制Delta-Vif FIV FIV，但对Delta-Bet-Bet Formy Formy病毒和FELV没有影响。 d。读取的替代剪接产生杂交两个催化结构域A3CH分子，使用隐形剪接供体位点与A3CC的TGA终止密码子重叠，并将A3H的Exon 2与A3H的Exon 2一起，并在该混合分子的A3H部分中转换。该A3CH分子具有针对Delta-VIF FIV和FELV的强大抗病毒活性，而不是针对Delta-Bet偏式病毒。 e。通过对九种猫品种的SNP分析，发现了A3C基因中非同义的替代。这些氨基酸位置位于涉及四聚体和二聚体形成的位点上。发现这些站点之一是确定人类和非洲绿色猴子APOBEC3G和VIF在HIV和Sivagm中的宿主特定相互作用的同一地点。现在，计划将部位直接诱变检查这些氨基酸取代对FIV，FELV和方向性病毒感染的影响。