细胞自噬在新城疫病毒感染过程中的作用
摘要
新城疫（Newcastle disease, ND）是一种由新城疫病毒（Newcastle disease virus, NDV）感染导致的高度接触性传染病。NDV能够感染多达240多种禽类，强毒NDV能够引起呼吸道、神经系统以及消化道的严重病变。NDV是单股不分节段的负股RNA病毒，隶属于副粘病毒科（Paramyxoviridae）禽腮腺炎病毒属（Avulavirus）。副粘病毒科其他成员例如麻疹病毒、尼帕病毒、亨德拉病毒和腮腺炎病毒能够感染多数哺乳动物甚至人并引起严重的疾病。尽管近年来反向遗传学技术的发展给我们对NDV的致病机理研究带来了很大便利，但病毒作为一种专性寄生性生物，在宿主体内的复制很大程度上决定于宿主生物对其的抵抗性以及病毒的逃逸机制，这就促使我们更多的从宿主角度来重新看待病毒的致病过程。
自噬（Autophagy）是进化学上保守的一种细胞内膜运输的过程，是细胞内物质成分转运至溶酶体被降解过程的统称，主要通过降解细胞中损坏的或不需要的成分来负责压力条件下细胞内环境的稳态。正常情况下，细胞自噬的基础水平维持在较低的水平以维持细胞内稳态，当细胞处于某些应激状态下，例如心脏病、肿瘤和传染病发生过程中，自噬水平迅速升高，通过细胞内组分的代谢提供能量和大分子物质来度过“危机”。由于进化中的保守型，自噬能够作为机体的固有防御机制发挥对病毒的抵抗性。另一方面，病毒作为专性细胞内寄生物，在长期与细胞自噬这种保守的细胞内膜运输过程互相影响的进化过程中，不但具备了逃逸宿主细胞自噬的功能，甚至能够利用自噬来促进自身复制。目前已有很多关于病毒和自噬相互作用的报道，为了探究NDV在禽源细胞中如何诱导自噬以及自噬对NDV的复制起如何作用，我们开展了如下研究：
1）NDV能够在禽源细胞中诱导自噬稳态的产生。为了观察NDV感染禽源细胞后能否诱导细胞自噬，我们用NDV感染禽源成纤维细胞系DF-1后，以透射电镜，激光共聚焦和Western-blot方法鉴定自噬稳态的产生，结果表明：NDV Herts/33株感染DF-1细胞后12-24h能在细胞中发现大量双层或单层膜结构；转染的GFP-LC3质粒在病毒感染后呈现显著的点状分布，尤其在NDV感染形成的合胞体中明显；LC3B的Western-blot结果显示病毒感染后期LC3-I向LC3-II的转化，但以紫外灭活病毒则不能诱导LC3的转化。同时我们还以另一种NDV毒株F48E8和原代成纤维细胞CEF进行验证，以上结果均显示NDV感染能够诱导自噬稳态的产生。
2）NDV能够在禽源细胞中诱导自噬流的产生。评价自噬水平的高低不仅仅指自噬体的形成，更重要的是整个自噬系统的流动水平的高低，即稳态自噬水平的增高有可能是自噬起始的增加亦或是自噬降解的减少两方面结果。为了验证NDV感染能否诱导完整自噬的产生，我们在NDV感染之后检测了自噬流标志蛋白p62的降解，溶酶体抑制剂E64d和pepstatinA处理之后LC3-II的变化以及GFP-LC3荧光的转移。结果表明NDV感染能够诱导p62蛋白的降解，同时以溶酶体抑制剂处理之后观察到LC3-II的显著增加，共聚焦结果显示GFP-LC3在病毒感染中后期发生向溶酶体的转移。以上结果均显示NDV感染能够诱导完整自噬流的产生。
3）通过药物或自噬相关基因的干扰将自噬阻断能够影响NDV的复制。为了进一步探究自噬对NDV复制的影响，我们首先利用自噬抑制药物渥曼青霉素和氯喹处理DF-1细胞，发现药物阻断自噬之后细胞中的病毒蛋白和细胞上清中的病毒滴度都有所降低，并且这种降低呈现药物依赖性。为了排除药物的非特异性，我们将自噬关键基因Beclin1进行干扰，发现Beclin1的干扰降低病毒的复制。同时我们以NDV感染自噬关键基因Atg5knockout鼠成纤维细胞系（MEFs）和野生型MEFs，发现Atg5的敲除显著降低了NDV的复制。以上结果显示自噬在NDV复制过程中起到了有利的作用。
4）NDV的结构蛋白与自噬相关蛋白互作。为了进一步从机理上解释NDV与自噬的直接关系，我们利用激光共聚焦和免疫共沉淀（Co-IP）的方法对NDV结构蛋白NP, P, M和自噬相关蛋白Beclin1和LC3B进行互作研究，结果显示NDV结构蛋白NP, M能够与Beclin1互作，而M还能与LC3B互作，这些结果显示自噬与NDV的复制直接相关。
5）体外实验结果证明NDV能够在DF-1细胞系上诱导自噬，自噬对病毒复制有利，为了验证体外结果，我们进行了以下体内实验，首先以NDV Herts/33感染1周龄SPF鸡，发现其能够在多种脏器包括：心、肝、脾和肺中诱导LC3的转化。而用自噬抑制药物处理能够降低肺中病毒滴度，减轻肺和肠道的病变，且渥曼青霉素的使用能为1周龄SPF鸡提供40%的保护率。以上实验结果显示NDV在体内也能诱导自噬产生，且自噬对病毒复制有利。
本论文以禽细胞为模式细胞证明了细胞自噬在NDV的复制中发挥重要的作用，更重要的是，我们首次以禽为模式生物研究体内状态下自噬与病毒的关系，这为进一步研究自噬与禽源病毒互作及开发新型抗病毒药物提供了线索。
Newcastle disease （ND） is a highly contagious infectious disease caused by Newcastle diseasevirus (NDV), which could infect more than240kinds of birds. Virulent NDV strains could inducesevere pathological changes in respiratory tracts, nervous systems and digestive tracts. NDV isclassified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae. Someother paramyxoviruses such as measles virus, Nipah virus, Hendra virus, and mumps virus areresponsible for a range of diseases in mammalian species including humans. Although the reversegenetic technique offers us great convenience to study the pathogenic mechanism of NDV infection, asobligate parasitic organisms, virus replication in host cells largely depends on the host resistance andvirus evading strategy, which leads us to consider more about the impact of host to the pathogenicprocess of virus.
Autophagy is an evolutionally conserved process of cellular inner membrane transportation, whichis responsible for transporting cellular elements to lysosomes for degradation. Autophagy degradesdamaged or unnecessary elements to maintain the steady-state intracellular environment under pressure.Under normal circumstances, cellular autophagy maintains at a low level to maintain steady-stateintracellular environment. When the cell is under some stress conditions, such as heart attack, tumer andinfection, the level of autophagy was elevated rapidly to go through the crisis. Autophagy functions asthe innate antiviral mechanism as it’s evolutionally conserved. On the other hand, as the obligateintracellular parasite, virus evades from the antiviral mechanism of autophagy, or even utilizesautophagy for its own replication in the long-term history. The last few years have seen a variety ofviruses which are in close relationship with autophagy. Our study focuses on how NDV inducesautophagy and whether NDV infection was influenced by autophagy.
To determine whether NDV infection induced autophagy, transmission electron microscopy（TEM）, was performed on DF-1cell line infected by NDV strain Herts/33. The results showed thatcompared with mock-infected cells, both double-or single-membrane vesicles were significantlyincreased post infection. To further verify NDV infection could activate the machinery of autophagy,GFP-LC3dot formation during NDV infection was investigated. Punctuate GFP-LC3proteinssignificantly accumulated in Herts/33infected cells, especially virus induced syncytia. The conversionfrom endogenous LC3-I to LC3-II, which could be detected by immunoblotting, is another convincingapproach to evaluate autophagy. DF-1cells infected by Herts/33presented significant LC3-I/LC3-IIconversion at late stages of infection. The conversion of LC3-I/LC3-II was largely undetectable at allthe time points post UV-inactivated Herts/33infection. Another NDV strain F48E8and another cellCEF were introduced and similar results was observed. Collectively, these results suggest steaty-stateautophagy was induced in NDV-infected chicken cells and the replication of NDV was essential forautophagy induction.
Autophagy not only refers to the formation of autophagosomes, but most importantly flux throughthe entire system including lysosomes, which means up-regulated level of steady-state autophagy may represent a reduced autophagic degradation rather than enhancement of the autophagic flux. Here weinvestigated p62degradation in the course of NDV infection. Infection of Herts/33caused thedegradation of p62in DF-1cells with the passage of time. Next we measured turnover of LC3-II in thepresence and absence of lysosomal protease inhibitors. Employment of both E64d and pepstatin Asignificantly increased the level of LC3-II, and p62in NDV-infected cells. Moreover, we observed thetranslocation of GFP-LC3from autophagosome to lysosome. These data clearly suggest that NDVinfection enhanced autophagic flux in DF-1and CEF cells.
To analysis the effect of autophagy on the replication of NDV in chicken cells, wortmannin andCQ, which could inhibit autophagy in different stages, was utilized to test their effect to virusreplication. The results showed that pharmacological inhibition of autophagy reduced NDV replicationin DF-1cells. Moreover knockdown of beclin1and knockout of atg5gene both decreased virusreplication. As a whole, our results strongly supported that autophagy machinery was required foreffective infection of NDV.
To further identify the direct interactin between virus and autophagy, next, we performed confocalmicroscopy and co-immunoprecipitation to confirm the direct interaction between NDV Nucleoproteinand Matrix protein and Beclin1. Moreover, NDV Matrix protein also binds to LC3B protein.
Given that autophagy was essential for NDV replication in vitro, we further studied the role ofautophagy in vivo after NDV infection. First we found NDV triggers LC3-I/LC3-II conversion invarious tissues of1-week-old chickens. Moreover, administration of autophagy inhibitor decreasedNDV production and pathogenesis in both lung and intestine. Moreover, administration of wortmanninincreased the survival rate of chickens. Together, these in vivo results further confirmed the studies invitro, which concluded that autophagy was critical for NDV infection.
In conclusion, we confirmed that autophagy was critical for NDV infection in vitro and in vivo.More importantly, for the first time, we used the chicken model to study autophagy and confirmed thatautophagy was critical for NDV infection. Our studies provided insights into virus-host interaction andthe development of antiviral strategies against avain virus infection.
自噬（Autophagy）是进化学上保守的一种细胞内膜运输的过程，是细胞内物质成分转运至溶酶体被降解过程的统称，主要通过降解细胞中损坏的或不需要的成分来负责压力条件下细胞内环境的稳态。正常情况下，细胞自噬的基础水平维持在较低的水平以维持细胞内稳态，当细胞处于某些应激状态下，例如心脏病、肿瘤和传染病发生过程中，自噬水平迅速升高，通过细胞内组分的代谢提供能量和大分子物质来度过“危机”。由于进化中的保守型，自噬能够作为机体的固有防御机制发挥对病毒的抵抗性。另一方面，病毒作为专性细胞内寄生物，在长期与细胞自噬这种保守的细胞内膜运输过程互相影响的进化过程中，不但具备了逃逸宿主细胞自噬的功能，甚至能够利用自噬来促进自身复制。目前已有很多关于病毒和自噬相互作用的报道，为了探究NDV在禽源细胞中如何诱导自噬以及自噬对NDV的复制起如何作用，我们开展了如下研究：
1）NDV能够在禽源细胞中诱导自噬稳态的产生。为了观察NDV感染禽源细胞后能否诱导细胞自噬，我们用NDV感染禽源成纤维细胞系DF-1后，以透射电镜，激光共聚焦和Western-blot方法鉴定自噬稳态的产生，结果表明：NDV Herts/33株感染DF-1细胞后12-24h能在细胞中发现大量双层或单层膜结构；转染的GFP-LC3质粒在病毒感染后呈现显著的点状分布，尤其在NDV感染形成的合胞体中明显；LC3B的Western-blot结果显示病毒感染后期LC3-I向LC3-II的转化，但以紫外灭活病毒则不能诱导LC3的转化。同时我们还以另一种NDV毒株F48E8和原代成纤维细胞CEF进行验证，以上结果均显示NDV感染能够诱导自噬稳态的产生。
2）NDV能够在禽源细胞中诱导自噬流的产生。评价自噬水平的高低不仅仅指自噬体的形成，更重要的是整个自噬系统的流动水平的高低，即稳态自噬水平的增高有可能是自噬起始的增加亦或是自噬降解的减少两方面结果。为了验证NDV感染能否诱导完整自噬的产生，我们在NDV感染之后检测了自噬流标志蛋白p62的降解，溶酶体抑制剂E64d和pepstatinA处理之后LC3-II的变化以及GFP-LC3荧光的转移。结果表明NDV感染能够诱导p62蛋白的降解，同时以溶酶体抑制剂处理之后观察到LC3-II的显著增加，共聚焦结果显示GFP-LC3在病毒感染中后期发生向溶酶体的转移。以上结果均显示NDV感染能够诱导完整自噬流的产生。
3）通过药物或自噬相关基因的干扰将自噬阻断能够影响NDV的复制。为了进一步探究自噬对NDV复制的影响，我们首先利用自噬抑制药物渥曼青霉素和氯喹处理DF-1细胞，发现药物阻断自噬之后细胞中的病毒蛋白和细胞上清中的病毒滴度都有所降低，并且这种降低呈现药物依赖性。为了排除药物的非特异性，我们将自噬关键基因Beclin1进行干扰，发现Beclin1的干扰降低病毒的复制。同时我们以NDV感染自噬关键基因Atg5knockout鼠成纤维细胞系（MEFs）和野生型MEFs，发现Atg5的敲除显著降低了NDV的复制。以上结果显示自噬在NDV复制过程中起到了有利的作用。
4）NDV的结构蛋白与自噬相关蛋白互作。为了进一步从机理上解释NDV与自噬的直接关系，我们利用激光共聚焦和免疫共沉淀（Co-IP）的方法对NDV结构蛋白NP, P, M和自噬相关蛋白Beclin1和LC3B进行互作研究，结果显示NDV结构蛋白NP, M能够与Beclin1互作，而M还能与LC3B互作，这些结果显示自噬与NDV的复制直接相关。
5）体外实验结果证明NDV能够在DF-1细胞系上诱导自噬，自噬对病毒复制有利，为了验证体外结果，我们进行了以下体内实验，首先以NDV Herts/33感染1周龄SPF鸡，发现其能够在多种脏器包括：心、肝、脾和肺中诱导LC3的转化。而用自噬抑制药物处理能够降低肺中病毒滴度，减轻肺和肠道的病变，且渥曼青霉素的使用能为1周龄SPF鸡提供40%的保护率。以上实验结果显示NDV在体内也能诱导自噬产生，且自噬对病毒复制有利。
本论文以禽细胞为模式细胞证明了细胞自噬在NDV的复制中发挥重要的作用，更重要的是，我们首次以禽为模式生物研究体内状态下自噬与病毒的关系，这为进一步研究自噬与禽源病毒互作及开发新型抗病毒药物提供了线索。
Newcastle disease （ND） is a highly contagious infectious disease caused by Newcastle diseasevirus (NDV), which could infect more than240kinds of birds. Virulent NDV strains could inducesevere pathological changes in respiratory tracts, nervous systems and digestive tracts. NDV isclassified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae. Someother paramyxoviruses such as measles virus, Nipah virus, Hendra virus, and mumps virus areresponsible for a range of diseases in mammalian species including humans. Although the reversegenetic technique offers us great convenience to study the pathogenic mechanism of NDV infection, asobligate parasitic organisms, virus replication in host cells largely depends on the host resistance andvirus evading strategy, which leads us to consider more about the impact of host to the pathogenicprocess of virus.
Autophagy is an evolutionally conserved process of cellular inner membrane transportation, whichis responsible for transporting cellular elements to lysosomes for degradation. Autophagy degradesdamaged or unnecessary elements to maintain the steady-state intracellular environment under pressure.Under normal circumstances, cellular autophagy maintains at a low level to maintain steady-stateintracellular environment. When the cell is under some stress conditions, such as heart attack, tumer andinfection, the level of autophagy was elevated rapidly to go through the crisis. Autophagy functions asthe innate antiviral mechanism as it’s evolutionally conserved. On the other hand, as the obligateintracellular parasite, virus evades from the antiviral mechanism of autophagy, or even utilizesautophagy for its own replication in the long-term history. The last few years have seen a variety ofviruses which are in close relationship with autophagy. Our study focuses on how NDV inducesautophagy and whether NDV infection was influenced by autophagy.
To determine whether NDV infection induced autophagy, transmission electron microscopy（TEM）, was performed on DF-1cell line infected by NDV strain Herts/33. The results showed thatcompared with mock-infected cells, both double-or single-membrane vesicles were significantlyincreased post infection. To further verify NDV infection could activate the machinery of autophagy,GFP-LC3dot formation during NDV infection was investigated. Punctuate GFP-LC3proteinssignificantly accumulated in Herts/33infected cells, especially virus induced syncytia. The conversionfrom endogenous LC3-I to LC3-II, which could be detected by immunoblotting, is another convincingapproach to evaluate autophagy. DF-1cells infected by Herts/33presented significant LC3-I/LC3-IIconversion at late stages of infection. The conversion of LC3-I/LC3-II was largely undetectable at allthe time points post UV-inactivated Herts/33infection. Another NDV strain F48E8and another cellCEF were introduced and similar results was observed. Collectively, these results suggest steaty-stateautophagy was induced in NDV-infected chicken cells and the replication of NDV was essential forautophagy induction.
Autophagy not only refers to the formation of autophagosomes, but most importantly flux throughthe entire system including lysosomes, which means up-regulated level of steady-state autophagy may represent a reduced autophagic degradation rather than enhancement of the autophagic flux. Here weinvestigated p62degradation in the course of NDV infection. Infection of Herts/33caused thedegradation of p62in DF-1cells with the passage of time. Next we measured turnover of LC3-II in thepresence and absence of lysosomal protease inhibitors. Employment of both E64d and pepstatin Asignificantly increased the level of LC3-II, and p62in NDV-infected cells. Moreover, we observed thetranslocation of GFP-LC3from autophagosome to lysosome. These data clearly suggest that NDVinfection enhanced autophagic flux in DF-1and CEF cells.
To analysis the effect of autophagy on the replication of NDV in chicken cells, wortmannin andCQ, which could inhibit autophagy in different stages, was utilized to test their effect to virusreplication. The results showed that pharmacological inhibition of autophagy reduced NDV replicationin DF-1cells. Moreover knockdown of beclin1and knockout of atg5gene both decreased virusreplication. As a whole, our results strongly supported that autophagy machinery was required foreffective infection of NDV.
To further identify the direct interactin between virus and autophagy, next, we performed confocalmicroscopy and co-immunoprecipitation to confirm the direct interaction between NDV Nucleoproteinand Matrix protein and Beclin1. Moreover, NDV Matrix protein also binds to LC3B protein.
Given that autophagy was essential for NDV replication in vitro, we further studied the role ofautophagy in vivo after NDV infection. First we found NDV triggers LC3-I/LC3-II conversion invarious tissues of1-week-old chickens. Moreover, administration of autophagy inhibitor decreasedNDV production and pathogenesis in both lung and intestine. Moreover, administration of wortmanninincreased the survival rate of chickens. Together, these in vivo results further confirmed the studies invitro, which concluded that autophagy was critical for NDV infection.
In conclusion, we confirmed that autophagy was critical for NDV infection in vitro and in vivo.More importantly, for the first time, we used the chicken model to study autophagy and confirmed thatautophagy was critical for NDV infection. Our studies provided insights into virus-host interaction andthe development of antiviral strategies against avain virus infection.
引文
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17Espert L, Denizot M, Grimaldi M, et al. Autophagy is involved in t cell death after binding of hiv-1envelope proteins to cxcr4[J]. J Clin Invest,2006,116(8):2161-2172.
18Fukuchi K, Watanabe H, Tomoyasu S, et al. Phosphatidylinositol3-kinase inhibitors, wortmanninor ly294002, inhibited accumulation of p21protein after gamma-irradiation by stabilization of theprotein [J]. Biochim Biophys Acta,2000,1496(2-3):207-220.
19Gannage M, Dormann D, Albrecht R, et al. Matrix protein2of influenza a virus blocksautophagosome fusion with lysosomes [J]. Cell Host Microbe,2009,6(4):367-380.
20Gannage M, Ramer PC, Munz C. Targeting beclin1for viral subversion of macroautophagy [J].Autophagy,2010,6(1):166-167.
21Garcia MA, Gil J, Ventoso I, et al. Impact of protein kinase pkr in cell biology: From antiviral toantiproliferative action [J]. Microbiol Mol Biol Rev,2006,70(4):1032-1060.
22Gosert R, Kanjanahaluethai A, Egger D, et al. Rna replication of mouse hepatitis virus takes placeat double-membrane vesicles [J]. J Virol,2002,76(8):3697-3708.
23Guevin C, Manna D, Belanger C, et al. Autophagy protein atg5interacts transiently with thehepatitis c virus rna polymerase (ns5b) early during infection [J]. Virology,2010,405(1):1-7.
24He B. Viruses, endoplasmic reticulum stress, and interferon responses [J]. Cell Death Differ,2006,13(3):393-403.
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26Horvath CM, Paterson RG, Shaughnessy MA, et al. Biological activity of paramyxovirus fusionproteins: Factors influencing formation of syncytia [J]. J Virol,1992,66(7):4564-4569.
27Huang SC, Chang CL, Wang PS, et al. Enterovirus71-induced autophagy detected in vitro and invivo promotes viral replication [J]. J Med Virol,2009,81(7):1241-1252.
28Isler JA, Skalet AH, Alwine JC. Human cytomegalovirus infection activates and regulates theunfolded protein response [J]. J Virol,2005,79(11):6890-6899.
29Itakura E, Kishi C, Inoue K, et al. Beclin1forms two distinct phosphatidylinositol3-kinasecomplexes with mammalian atg14and uvrag [J]. Mol Biol Cell,2008,19(12):5360-5372.
30Jackson WT, Giddings TH, Jr., Taylor MP, et al. Subversion of cellular autophagosomal machineryby rna viruses [J]. PLoS Biol,2005,3(5):e156.
31Joubert PE, Meiffren G, Gregoire IP, et al. Autophagy induction by the pathogen receptor cd46[J].Cell Host Microbe,2009,6(4):354-366.
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33Kabeya Y, Mizushima N, Ueno T, et al. Lc3, a mammalian homologue of yeast apg8p, is localizedin autophagosome membranes after processing [J]. EMBO J,2000,19(21):5720-5728.
34Kamada Y, Funakoshi T, Shintani T, et al. Tor-mediated induction of autophagy via an apg1proteinkinase complex [J]. J Cell Biol,2000,150(6):1507-1513.
35Karim MR, Kanazawa T, Daigaku Y, et al. Cytosolic lc3ratio as a sensitive index ofmacroautophagy in isolated rat hepatocytes and h4-ii-e cells [J]. Autophagy,2007,3(6):553-560.
36Khakpoor A, Panyasrivanit M, Wikan N, et al. A role for autophagolysosomes in dengue virus3production in hepg2cells [J]. J Gen Virol,2009,90(Pt5):1093-1103.
37Kihara A, Noda T, Ishihara N, et al. Two distinct vps34phosphatidylinositol3-kinase complexesfunction in autophagy and carboxypeptidase y sorting in saccharomyces cerevisiae [J]. J Cell Biol,2001,152(3):519-530.
38Kim HJ, Lee S, Jung JU. When autophagy meets viruses: A double-edged sword with functions indefense and offense [J]. Semin Immunopathol,2010,32(4):323-341.
39Kim J, Huang WP, Stromhaug PE, et al. Convergence of multiple autophagy and cytoplasm tovacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicleformation [J]. J Biol Chem,2002,277(1):763-773.
40Klionsky DJ, Abeliovich H, Agostinis P, et al. Guidelines for the use and interpretation of assaysfor monitoring autophagy in higher eukaryotes [J]. Autophagy,2008,4(2):151-175.
41Klionsky DJ, Abdalla FC, Abeliovich H, et al. Guidelines for the use and interpretation of assaysfor monitoring autophagy [J]. Autophagy,2012,8(4):445-544.
42Komatsu M, Wang QJ, Holstein GR, et al. Essential role for autophagy protein atg7in themaintenance of axonal homeostasis and the prevention of axonal degeneration [J]. Proc Natl AcadSci U S A,2007,104(36):14489-14494.
43Kong D, Yamori T. Phosphatidylinositol3-kinase inhibitors: Promising drug candidates for cancertherapy [J]. Cancer Sci,2008,99(9):1734-1740.
44Korenaga M, Ando M, Nishina S, et al. Replication of a full-length hepatitis c virus genomeinhibits mitochondrial complex i activity and increases mitochondrial ros production in cell culturesystem [J]. Hepatology,2006,44(4):294a-294a.
45Knight ZA, Shokat KM. Chemically targeting the pi3k family [J]. Biochem Soc Trans,2007,35(Pt2):245-249.
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52Lee YR, Lei HY, Liu MT, et al. Autophagic machinery activated by dengue virus enhances virusreplication [J]. Virology,2008,374(2):240-248.
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18Fukuchi K, Watanabe H, Tomoyasu S, et al. Phosphatidylinositol3-kinase inhibitors, wortmanninor ly294002, inhibited accumulation of p21protein after gamma-irradiation by stabilization of theprotein [J]. Biochim Biophys Acta,2000,1496(2-3):207-220.
19Gannage M, Dormann D, Albrecht R, et al. Matrix protein2of influenza a virus blocksautophagosome fusion with lysosomes [J]. Cell Host Microbe,2009,6(4):367-380.
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26Horvath CM, Paterson RG, Shaughnessy MA, et al. Biological activity of paramyxovirus fusionproteins: Factors influencing formation of syncytia [J]. J Virol,1992,66(7):4564-4569.
27Huang SC, Chang CL, Wang PS, et al. Enterovirus71-induced autophagy detected in vitro and invivo promotes viral replication [J]. J Med Virol,2009,81(7):1241-1252.
28Isler JA, Skalet AH, Alwine JC. Human cytomegalovirus infection activates and regulates theunfolded protein response [J]. J Virol,2005,79(11):6890-6899.
29Itakura E, Kishi C, Inoue K, et al. Beclin1forms two distinct phosphatidylinositol3-kinasecomplexes with mammalian atg14and uvrag [J]. Mol Biol Cell,2008,19(12):5360-5372.
30Jackson WT, Giddings TH, Jr., Taylor MP, et al. Subversion of cellular autophagosomal machineryby rna viruses [J]. PLoS Biol,2005,3(5):e156.
31Joubert PE, Meiffren G, Gregoire IP, et al. Autophagy induction by the pathogen receptor cd46[J].Cell Host Microbe,2009,6(4):354-366.
32Jounai N, Takeshita F, Kobiyama K, et al. The atg5atg12conjugate associates with innate antiviralimmune responses [J]. Proc Natl Acad Sci U S A,2007,104(35):14050-14055.
33Kabeya Y, Mizushima N, Ueno T, et al. Lc3, a mammalian homologue of yeast apg8p, is localizedin autophagosome membranes after processing [J]. EMBO J,2000,19(21):5720-5728.
34Kamada Y, Funakoshi T, Shintani T, et al. Tor-mediated induction of autophagy via an apg1proteinkinase complex [J]. J Cell Biol,2000,150(6):1507-1513.
35Karim MR, Kanazawa T, Daigaku Y, et al. Cytosolic lc3ratio as a sensitive index ofmacroautophagy in isolated rat hepatocytes and h4-ii-e cells [J]. Autophagy,2007,3(6):553-560.
36Khakpoor A, Panyasrivanit M, Wikan N, et al. A role for autophagolysosomes in dengue virus3production in hepg2cells [J]. J Gen Virol,2009,90(Pt5):1093-1103.
37Kihara A, Noda T, Ishihara N, et al. Two distinct vps34phosphatidylinositol3-kinase complexesfunction in autophagy and carboxypeptidase y sorting in saccharomyces cerevisiae [J]. J Cell Biol,2001,152(3):519-530.
38Kim HJ, Lee S, Jung JU. When autophagy meets viruses: A double-edged sword with functions indefense and offense [J]. Semin Immunopathol,2010,32(4):323-341.
39Kim J, Huang WP, Stromhaug PE, et al. Convergence of multiple autophagy and cytoplasm tovacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicleformation [J]. J Biol Chem,2002,277(1):763-773.
40Klionsky DJ, Abeliovich H, Agostinis P, et al. Guidelines for the use and interpretation of assaysfor monitoring autophagy in higher eukaryotes [J]. Autophagy,2008,4(2):151-175.
41Klionsky DJ, Abdalla FC, Abeliovich H, et al. Guidelines for the use and interpretation of assaysfor monitoring autophagy [J]. Autophagy,2012,8(4):445-544.
42Komatsu M, Wang QJ, Holstein GR, et al. Essential role for autophagy protein atg7in themaintenance of axonal homeostasis and the prevention of axonal degeneration [J]. Proc Natl AcadSci U S A,2007,104(36):14489-14494.
43Kong D, Yamori T. Phosphatidylinositol3-kinase inhibitors: Promising drug candidates for cancertherapy [J]. Cancer Sci,2008,99(9):1734-1740.
44Korenaga M, Ando M, Nishina S, et al. Replication of a full-length hepatitis c virus genomeinhibits mitochondrial complex i activity and increases mitochondrial ros production in cell culturesystem [J]. Hepatology,2006,44(4):294a-294a.
45Knight ZA, Shokat KM. Chemically targeting the pi3k family [J]. Biochem Soc Trans,2007,35(Pt2):245-249.
46Ku B, Woo JS, Liang C, et al. Structural and biochemical bases for the inhibition of autophagy andapoptosis by viral bcl-2of murine gamma-herpesvirus68[J]. PLoS Pathog,2008,4(2):e25.
47Kudchodkar SB, Levine B. Viruses and autophagy [J]. Rev Med Virol,2009,19(6):359-378.
48Kudchodkar SB, Yu Y, Maguire TG, et al. Human cytomegalovirus infection inducesrapamycin-insensitive phosphorylation of downstream effectors of mtor kinase [J]. J Virol,2004,78(20):11030-11039.
49Kuma A, Hatano M, Matsui M, et al. The role of autophagy during the early neonatal starvationperiod [J]. Nature,2004,432(7020):1032-1036.
50Kyei GB, Dinkins C, Davis AS, et al. Autophagy pathway intersects with hiv-1biosynthesis andregulates viral yields in macrophages [J]. J Cell Biol,2009,186(2):255-268.
51Lee HK, Lund JM, Ramanathan B, et al. Autophagy-dependent viral recognition by plasmacytoiddendritic cells [J]. Science,2007,315(5817):1398-1401.
52Lee YR, Lei HY, Liu MT, et al. Autophagic machinery activated by dengue virus enhances virusreplication [J]. Virology,2008,374(2):240-248.
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