Abstract：
The Sf9 cell line, originally isolated from the ovarian tissue of Spodoptera frugiperda larvae, is widely used in academia and industry for the baculovirus-mediated production of recombinant proteins and virus-like particles. RNA interference (RNAi) is a conserved antiviral pathway present in eukaryotic organisms and is the primary antiviral defence mechanism in insects. Recent evidence has implicated RNAi as an antiviral response to baculovirus infection in Sf9 cells. To test this hypothesis, CRISPR/Cas9 technology was used to disable the RNAi pathway in Sf9 cells by knocking out Dicer-2, the protein responsible for cleaving viral double-stranded RNA precursors into short interfering RNAs. Infection of Dicer-2 knockout Sf9 cells with either the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), recombinant AcMNPV (rAcMNPV) expressing β-galactosidase (β-gal), or rAcMNPV expressing a wasp venom protein (Vn50) at a multiplicity of infection (m.o.i.) of 1 resulted in a modest increase in virus replication compared to control Sf9 cells under adherent culture conditions. In contrast, Dicer-2 knockout Sf9 monolayer or suspension cultures infected by the rAcMNPV expressing β-gal at higher m.o.i.s (3.5 and 20) did not exhibit increases in either viral DNA replication or β-gal production. Intriguingly, during long-term passaging in suspension, Dicer-2 knockout Sf9 cultures underwent transient crashes in cell proliferation and viability. It was discovered that these periods of low growth and viability coincided with a dramatic increase in the RNA levels of S. frugiperda rhabdovirus, a recently identified adventitious virus that persistently infects the Sf9 cell line, suggesting a role for Dicer-2 in managing chronic viral infections in this industrially relevant insect cell line.
摘要：
Sf9细胞系,最初从节食夜蛾幼虫的卵巢组织中分离出来，在学术界和工业界广泛用于杆状病毒介导的重组蛋白和病毒样颗粒的生产。RNA干扰(RNAi)是真核生物中存在的保守的抗病毒途径,并且是昆虫中的主要抗病毒防御机制。最近的证据表明RNAi是Sf9细胞中对杆状病毒感染的抗病毒反应。为了检验这个假设，CRISPR/Cas9技术用于通过敲除Dicer-2(负责将病毒双链RNA前体切割成短干扰RNA的蛋白质)来禁用Sf9细胞中的RNAi途径。用野生型杆状病毒（AcMNPV）感染Dicer-2敲除的Sf9细胞，表达β-半乳糖苷酶（β-gal）的重组AcMNPV（rAcMNPV），在贴壁培养条件下,与对照Sf9细胞相比,以1的感染复数(m.o.i.)表达黄蜂毒液蛋白(Vn50)的rAcMNPV导致病毒复制的适度增加。相比之下,被表达β-gal的rAcMNPV感染的Dicer-2敲除Sf9单层或悬浮培养物在较高的m.o.i.s（3.5和20）下没有表现出病毒DNA复制或β-gal产生的增加。有趣的是，在长期停药期间，Dicer-2敲除的Sf9培养物在细胞增殖和活力方面经历了短暂的崩溃。人们发现，这些低生长和活力时期与S.frugiperda弹状病毒的RNA水平急剧增加相吻合，一种最近发现的持续感染Sf9细胞系的不定病毒，提示Dicer-2在这种工业相关的昆虫细胞系中管理慢性病毒感染中的作用。
