腸病毒71型(EV-A71)是一種無外套模病毒，內含一股單鏈核糖核酸(RNA)包覆於四種結構莢膜蛋白(VP1~VP4)之中。EV-A71感染造成幼童手口足症或是中樞神經系統病變，也曾發現會在不同細胞株中引起粒線體相關的細胞凋亡(apoptosis)訊息及半胱氨酸蛋白酶(caspases)主導的細胞凋亡訊號路徑，是對抗病毒感染是一種很重要的細胞防禦機制。然而在感染時，病毒如何在時空點上利用病毒蛋白和細胞蛋白之間的運作來主導病毒蛋白生合成和細胞凋亡訊號的產生仍然有許多未知。實驗室先前利用合併的串聯親和性蛋白純化技術與質譜儀LC-MS/MS蛋白分析策略，發現了與VP4有交互作用的細胞蛋白網絡複合物。在 39種和VP4有交互作用的關係的宿主細胞蛋白中，receptor of the globular heads of C1q receptor (gC1qR)身份的可信度mascot 得分值最高，而且廣泛分布在細胞之中。我們的先前結果利用免疫共沉澱法及共軛焦顯微鏡影像系統，支持gC1qR和VP4之間親密的交互作用。因此在本研究我們進一步分析此gC1qR蛋白在引起EV-A71感染橫紋肌瘤(RD)細胞的凋亡訊息機制中所造成的影響，因為gC1qR已知會移進粒線體而調節細胞抗病毒防禦機制。首先我們的結果發現在病毒感染劑量為5MOI的狀況之下，內生性gC1qR會和緩的上升並且在感染後4小時開始大量出現病毒結構蛋白，同時也會引起caspase-3活化態增加的現象。其次，減抑gC1qR 基因表達(knockdown)會促使細胞內的病毒VPs產量增加，以及細胞內膜的磷脂絲胺酸(phosphatidyserine, PS)翻轉到細胞外膜的量增加引發細胞凋亡。宿主內的粒腺體膜電位也在較早期便發生變異，這樣的膜電位變異可能是由於病毒蛋白轉移到粒線體之中所導致，甚而引起下游的caspase-3和caspase-9的活化，促使細胞凋亡而抑止了感染進程。這些結果顯示腸病毒感染時gC1qR具有拮抗病毒生成及細胞凋亡的功能。也確實如此，當我們過度表達gC1qR (overexpression)，會抑制細胞內的病毒蛋白產成，也會抑制PS蛋白的翻轉。更甚者，過多的gC1qR會抑阻EV-A71感染所引起的膜電位變異，同時細胞質中的病毒蛋白也明顯的減少，也並沒有發現有過多比例的病毒蛋白轉移到粒線體中，且粒線體下游的細胞凋亡訊息路徑 caspase-3和caspase-9的活性也明顯下降。上述結果暗示著內生性gC1qR和VP4之間的交互作用會抑止細胞過早進行凋亡，並可能隨著感染時間愈行負向的調控病毒轉譯或促使病毒蛋白降解，需要再進一步深入探討。本研究初步發現gC1qR在EV-A71感染中隨時間而增加，可在早中期抑阻粒線體相關的細胞凋亡訊息及其下游訊息路徑，也能在中晚期負向參與調控病毒蛋白的生合成。

Enterovirus A71 (EV-A71) is a non-enveloped virus which contains a single-stranded positive RNA genome encapsidated by four viral structural proteins (VPs), VP1, VP2, VP3 and VP4. Infection of EV-A71 causes an array of childhood diseases with severe neurological manifestations and has been shown to induce mitochondrial-associated apoptotic signaling and caspase-mediated signaling events in many types of cells. Cellular apoptosis is a crucial cellular defense to account for EV-A71 invasion; however, it remains largely unclear the spatiotemporal control of viral and cellular factors that are utilized by invading EV-A71 for the establishment of productive infection versus the initiation of apoptosis. Our lab had previous identified a cellular interactome of VP4 by a combined tandem-tag affinity purification (TAP) technique and LC/MS-MS-based protein identification approach. Among the 39 VP4-interacting cellular candidates, receptor of the globular heads of C1q complement (gC1qR) shows the highest identification fidelity and is ubiquitously present in cells. Our previous finding supports intimate gC1qR-VP4 interaction and co-localization in cytoplasm and mitochondria in infected RD cells by immunoprecipitation and confocal imagines. In this study, we step further the role of gC1qR in mitochondria-associated apoptosis, because gC1qR is capable of entering mitochondria to modulate cellular defense machinery. Firstly, our results found that infection of RD cells with 5 MOI of EV-A71 resulted in increased qC1qR and viral structural protein during the productive phase of viral growth within 4-8 hours. The caspase-3 was also activated after 4 hours post-infection. EV-A71 infection of gC1qR-knockdown RD cells, in comparison to infected sh-control cells, promoted intracellular VPs production and numbers of apoptotic cells with flipped phosphatidylserine (PS) protein on the outer cell membrane. Moreover, VPs were found to translocate to the mitochondria which may cause mitochondrial stress, as mitochondrial membrane potential were lost quickly after infection, alone with increasing levels of cleaved forms of caspase-9 and caspase-3. These results suggested that gC1qR is required to negatively modulate EV-A71-induced apoptosis and productive viral production in RD cells. Indeed, EV-A71 infection of gC1qR-overexpressing RD cells, in comparison to infected RD cells, resulted in decreased VPs and membrane PS translocation. Also, gC1qR-overexpression protected the loss of MMP that occurred during EV-A71 infection, which accompanied with decreased VPs in the cytosolic and mitochondrial fraction, as well as decreased activation of caspase-9 and caspase-3. These results implied that endogenous gC1qR may interact with VP4 to retard early apoptosis and accumulate through time to negatively modulate intracellular VPs biogenesis via viral translation or cellular degradation machinery, yet the in-depth mechanism remains to be clarified. In conclusion, this study provides a preliminary evidence that gC1qR has a novel role during EV-A71 infection to spatiotemporally modulate cellular apoptosis. EV-A71-induced mitochondria-associated downstream apoptotic signaling can be retarded through overexpressing gC1qR, which is likely to be critical for EV-A71 to establish early- to mid-stage (4-6 h) of intracellular viral production, while accumulated gC1qR is capable of negative modulating viral protein production, which is likely to occur during mid- to late-stage (6-8 h) of infection when viral protein production is no longer needed.

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