ABSTRACT

Cellular metabolism caters to the energy and metabolite needs of cells. Although the role of the terminal metabolic enzyme LDHB (lactate dehydrogenase B) in the glycolysis pathway has been widely studied in cancer cells, its role in viral infection is relatively unknown. In this study, we found that CSFV (classical swine fever virus) infection reduces pyruvate levels while promotes lactate release in pigs and in PK-15 cells. Moreover, using a yeast two-hybrid screening system, we identified LDHB as a novel interacting partner of CSFV non-structural protein NS3. These results were confirmed via co-immunoprecipitation, glutathione S-transferase and confocal assays. Furthermore, knockdown of LDHB via interfering RNA induced mitochondrial fission and mitophagy, as detected reduced mitochondrial mass. Upon inhibition of LDHB, expression of the mitophagy proteins TOMM20 and VDAC1 decreased and the ubiquitination of MFN2, a mitochondrial fusion mediator, was promoted. In addition, a sensitive dual fluorescence reporter (mito-mRFP-EGFP) was utilized to analyze the delivery of autophagosomes to lysosomes in LDHB inhibition cells. Furthermore, LDHB inhibition promoted NFKB signaling, which was regulated by mitophagy; meanwhile, infection with CSFV negated these NFKB anti-viral responses. Inhibition of LDHB also inhibited apoptosis, providing an environment conducive to persistent viral infection. Finally, we demonstrated that LDHB inhibition promoted CSFV growth via mitophagy, whereas its overexpression decreased CSFV replication. Our data revealed a novel mechanism through which LDHB, a metabolic enzyme, mediates CSFV infection, and provides new avenues for the development of anti-viral strategies.Abbreviations: 3-MA:3-methyladenine; CCCP:carbonyl cyanide 3-chlorophenylhydrazone; CCK-8:cell counting kit-8; CSFV:classical swine fever virus; DAPI:4′,6-diamidino-2-phenylindole; DMSO:dimethyl sulfoxide; EGFP:enhanced green fluorescent protein; FBS:fetal bovine serum; FITC:fluorescein isothiocyanate; GST:glutathione-S-transferase; HCV:hepatitis C virus; IFN:interferon; LDH:lactate dehydrogenase; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MFN2:mitofusin 2; MOI:multiplicity of infection; NFKB:nuclear factor kappa B subunit 1; NFKBIA:nuclear factor inhibitor alpha; NS3:nonstructural protein 3; NKIRAS2:NFKB inhibitor interacting Ras like 2; PRKN:parkin E3 ubiquitin protein ligase; PBS:phosphate-buffered saline; qRT-PCR:real-time quantitative reverse transcriptase polymerase chain reaction; RELA:RELA proto-oncogene, NF-kB subunit; shRNA: short hairpin RNA; siRNA: small interfering RNA; TCID50:50% tissue culture infectious doses; TEM:transmission electron microscopy; TNF:tumor necrosis factor; TOMM20:translocase of outer mitochondrial membrane 20; VDAC1:voltage dependent anion channel 1.

摘要

细胞代谢迎合细胞的能量和代谢物需求。尽管末端代谢酶 LDHB（乳酸脱氢酶 B）在糖酵解途径中的作用已在癌细胞中得到广泛研究，但其在病毒感染中的作用相对未知。在这项研究中，我们发现 CSFV（经典猪瘟病毒）感染会降低丙酮酸水平，同时促进猪和 PK-15 细胞中的乳酸释放。此外，使用酵母双杂交筛选系统，我们将 LDHB 鉴定为 CSFV 非结构蛋白 NS3 的新型相互作用伙伴。这些结果通过免疫共沉淀、谷胱甘肽 S-转移酶和共聚焦测定得到证实。此外，LDHB的敲低通过干扰 RNA 诱导线粒体分裂和线粒体自噬，检测到线粒体质量减少。抑制 LDHB 后，线粒体自噬蛋白 TOMM20 和 VDAC1 的表达降低，线粒体融合介质 MFN2 的泛素化得到促进。此外，利用敏感的双荧光报告器（mito-mRFP-EGFP）分析自噬体向 LDHB 抑制细胞中溶酶体的传递。此外，LDHB 抑制促进了受线粒体自噬调节的 NFKB 信号传导；同时，感染 CSFV 会抵消这些 NFKB 抗病毒反应。抑制 LDHB 也抑制细胞凋亡，提供有利于持续病毒感染的环境。最后，我们证明了 LDHB 抑制通过线粒体自噬促进了 CSFV 的生长，而其过表达降低了 CSFV 的复制。缩写：3-MA:3-甲基腺嘌呤；CCCP：羰基氰化物3-氯苯腙；CCK-8：细胞计数试剂盒-8；CSFV：经典猪瘟病毒；DAPI：4',6-二脒基-2-苯基吲哚；DMSO：二甲基亚砜；EGFP：增强型绿色荧光蛋白；FBS：胎牛血清；FITC：异硫氰酸荧光素；GST：谷胱甘肽-S-转移酶；HCV：丙型肝炎病毒；干扰素：干扰素；LDH：乳酸脱氢酶；MAP1LC3/LC3：微管相关蛋白1轻链3；MFN2：mitofusin 2；MOI：感染复数；NFKB：核因子 kappa B 亚基 1；NFKBIA：核因子抑制剂α；NS3：非结构蛋白 3；NKIRAS2：NFKB 抑制剂与 Ras 样 2 相互作用；PRKN：parkin E3泛素蛋白连接酶；PBS：磷酸盐缓冲盐水；qRT-PCR：实时定量逆转录酶聚合酶链反应；RELA：RELA原癌基因，NF-kB亚基；shRNA：短发夹RNA；siRNA：小干扰RNA；TCID50：50% 组织培养感染剂量；TEM：透射电子显微镜；TNF：肿瘤坏死因子；TOMM20：线粒体外膜转位酶 20；VDAC1：电压依赖性阴离子通道 1。