鼠白血病病毒glycosylated Gag及马传染性贫血病毒S2拮抗SERINC5抗病毒活性机制的研究

发布时间：2021-04-25 18:53

　　SERINC家族属于多重跨膜蛋白,这个家族包括5个成员,但各成员的功能目前并不清楚。其中SERINC5（Ser5）作为逆转录病毒限制因子,通过未知机制插入到病毒粒子中并抑制病毒侵入下一个靶细胞。研究发现人免疫缺陷病毒（HIV-1）的附属蛋白Nef能将Ser5内化到细胞浆中并通过溶酶体降解Ser5,进而阻止Ser5进入病毒粒子。除了HIV-1 Nef蛋白之外,鼠白血病病毒（MLV）的附属蛋白glycoGag以及马传染性贫血病毒（EIAV）的附属蛋白S2也能拮抗Ser5对HIV-1病毒的限制作用。然而,MLV glycoGag以及EIAV S2拮抗Ser5的机制目前还不清楚。在本论文中,我们系统探讨了glycoGag及S2如何拮抗Ser5蛋白的抗病毒活性。我们研究发现Ser5能插入到MLV的病毒粒子中并限制其感染性,而glycoGag能阻止Ser5插入到病毒粒子中并拮抗Ser5的抗病毒活性。研究发现glycoMA,既GlycoGag蛋白N端的189个氨基酸能发挥和glycoGag一样的功能,因此我们利用glycoMA深入探讨了其拮抗Ser5的详细机制。我们发现glycoMA能将Ser5从细... 

【文章来源】：中国农业科学院北京市

【文章页数】：95 页

【学位级别】：博士

【文章目录】：
摘要
abstract
Abbreviations
CHAPTER 1 INTRODUCTION
    1.1 Restriction factors
        1.1.1 Discovery and molecular biology of SERINC genes
        1.1.2 Identification of Ser5 and Ser3 as restriction factors for HIV-
        1.1.3 Ser5 mediated inhibition of viral infectivity
        1.1.4 Mechanistic understanding of viral fusion inhibition triggered by Ser
    1.2 Retroviruses
        1.2.1 HIV-1
        1.2.2 MLV glycoGag
        1.2.3 EIAV S2
    1.3 Aims of the project
CHAPTER 2 MURINE LEUKEMIA VIRUS ACCESSORY PROTEINS GLYCOGAG REDUCES MURINE SERINC5 PROTEIN EXPRESSION LEVEL VIA DEGRADING LYSOSOMAL PATHWAY TO COUNTERACT SERINC5 ANTIRETROVIRAL ACTIVITY
    2.1 Background
    2.2 Material and methods
        2.2.1 Cells and culturing system
        2.2.2 Plasmids
        2.2.3 Transfection
        2.2.4 Effect of mSer5 on HIV-1 infectivity
        2.2.5 Effect of mSer5 on MLV infectivity
        2.2.6 Effect of glycoGag on Ser5 endocytosis
        2.2.7 Western blotting
        2.2.8 Confocal microscopy
        2.2.9 Co-immunoprecipitation
        2.2.10 Flow cytometry
        2.2.11 Statistical analysis
    2.3 Results
        2.3.1 Plasmids confirmation
        2.3.2 Ser5 restricts MLV infectivity
        2.3.3 Ser5 incorporates into MLV particles
        2.3.4 GlycoMA and Nef counteracts Ser5 restriction and restore HIV-1 infectivity
        2.3.5 GlycoMA downregulates Ser5 expression
        2.3.6 GlycoMA re-localizes Ser5 to cytoplasmic compartments
        2.3.7 GlycoMA reduces Ser5 cell surface expression
        2.3.8 GlycoMA internalizes Ser5 via endocytosis
        2.3.9 GlycoMA utilizes Ap-2 pathway for Ser5 endocytosis
        2.3.10 Identification of crucial glycoGag residues responsible for Ser5 downregulation
        2.3.11 Intracellular interactions of glycoMA with Ser
        2.3.12 Rab GTPases are involved in Ser5 downregulation
        2.3.13 Ubiquitination plays crucial role in glycoMA-mediated Ser5 downregulation
        2.3.14 GlycoMA targets Ser5 to lysosomes for degradation
        2.3.15 GlycoMA downregulates murine Ser1,Ser2 and Ser
    2.4 Discussion
CHAPTER 3 MECHANISTIC INVESTIGATIONS OF SERINC5 COUNTERACTION TRIGGERED BY EQUINE INFECTIOUS ANEMIA VIRUS ACCESSORY PROTEIN S2
    3.1 Background
    3.2 Material and methods
        3.2.1 Cells and culturing system
        3.2.2 Plasmids
        3.2.3 Transfection
        3.2.4 Effect of S2 on HIV-1?N infectivity in the presence mSer
        3.2.5 Effect of S2 on Ser5 endocytosis
        3.2.6 Western blotting
        3.2.7 Confocal microscopy
        3.2.8 Flow cytometry
        3.2.9 Co-Immunoprecipitation
        3.2.10 Statistical analysis
    3.3 Results
        3.3.1 Plasmids confirmation
        3.3.2 S2 rescues HIV-1 infectivity and counteract Ser5 restriction
        3.3.3 S2 triggered Ser5 down-regulation
        3.3.4 S2 internalizes Ser5 to cytoplasmic compartments
        3.3.5 Crucial residues of S2 for interaction with Ser
        3.3.6 Intracellular interactions of S2 and Ser
        3.3.7 S2 internalizes Ser5 via endocytosis
        3.3.8 S2 utilizes AP-2 pathway for Ser5 internalization
        3.3.9 Rab GTPases are involved in Ser5 downregulation
        3.3.10 Ubiquitination plays crucial role in S2-mediated Ser5 downregulation
        3.3.11 S2 targets Ser5 to lysosomes for degradation
        3.3.12 Equine Ser5 restricts retroviral infectivity and counteracted by S
        3.3.13 S2 downregulates other members of SERINC family（Ser1,Ser2 and Ser3）
        3.3.14 S2 mediated downregulation of Ser5 is stronger than Nef
        3.3.15 S2 and glycoMA are broader antagonists of Ser5 than Nef
    3.4 Discussion
CHAPTER 4 CONCLUSIONS
REFERENCES
Curriculum Vitae



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