AAV Exploits Subcellular Stress Associated with Inflammation, Endoplasmic Reticulum Expansion, and Misfolded Proteins in Models of Cystic Fibrosis

Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency.

感染屏障通过多重层级发挥防御作用，可阻断病毒、细菌与寄生虫劫持宿主细胞以实现自身增殖的过程。一项颇具研究价值的假说指出，若细胞遭遇应激——如炎症、内质网（ER）扩张或错误折叠蛋白所引发的应激——则亚细胞屏障抵御病毒感染的效能将出现显著下降。本研究以囊性纤维化（CF）模型为实验体系，旨在探究亚细胞应激是否会提升腺相关病毒（AAV）的感染易感性。在气液界面培养的人气道上皮细胞模型中，我们利用源自CF患者肺部的黏液脓性物质的上清液，模拟了亚细胞应激与内质网扩张的生理病理状态。通过该炎症刺激物重现病变气道内的应激微环境，实验证实AAV的感染效率得到显著增强。由于超过90%的CF病例与囊性纤维化跨膜传导调节因子的错误折叠变体（ΔF508-CFTR）相关，我们进一步验证了错误折叠蛋白的存在是否会独立提升AAV的感染易感性。在上述模型中，表达ΔF508-CFTR的细胞的AAV转导效率较表达野生型CFTR的细胞高出一个数量级。在低温条件下对错误折叠的ΔF508-CFTR进行拯救后，病毒转导效率恢复至对照组水平，这表明与蛋白错误折叠相关的机制是提升感染易感性的核心原因。通过测试其他CFTR突变体G551D、D572N及1410X，我们证实该现象普遍存在于其他错误折叠蛋白介导的过程中，且与CFTR活性的丧失无关联。错误折叠蛋白的存在并未影响病毒的细胞表面附着，也未改变质粒转染实验中启动子-转基因盒的表达水平，这提示病毒的劫持作用发生在病毒粒子转运或加工层面。因此我们推测，在分泌通路中被招募以处理ΔF508-CFTR等错误折叠蛋白的因子，同时也发挥着限制病毒感染的作用。为验证这一假说，我们发现AAV会转运至微管组织中心，并定位于高尔基体/内质网转运蛋白附近区域。此外，通过小干扰RNA（siRNA）介导敲低参与ΔF508-CFTR加工或介导高尔基体-内质网逆行货物分选的蛋白（钙连蛋白、KDEL受体（KDEL-R）、β-COP及PSMB3），可有效调节AAV的感染效率。综上，本研究数据支持如下模型：AAV可利用受损的分泌系统完成感染过程；尤为重要的是，该研究凸显了遭遇内源性或外源性损伤的亚细胞应激环境，对病毒感染效率的影响程度之显著。