Targeted Deletion of Glycoprotein B Gene by CRISPR/Cas9 Nuclease Inhibits Gallid herpesvirus Type 3 in Dually Infected Marek's Disease Virus-Transformed Lymphoblastoid Cell Line MSB-1.
Targeted Deletion of Glycoprotein B Gene by CRISPR/Cas9 Nuclease Inhibits Gallid herpesvirus Type 3 in Dually Infected Marek's Disease Virus-Transformed Lymphoblastoid Cell Line MSB-1.
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DOI：
10.1128/jvi.02027-21
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发表时间：
2022-03-23
影响因子：
5.4
通讯作者：
Yao Y
中科院分区：
文献类型：
--
作者：
Zhang Y;Li W;Tang N;Moffat K;Nair V;Yao Y
马立克氏病病毒（Marek's disease virus，MDV）是α疱疹病毒亚科（Alphaherpesvirinae）中马病毒属（Mardivirus）的成员。MDV有三种不同的血清型，称为MDV-1（鸡疱疹病毒2型）、MDV-2（鸡疱疹病毒3型）和MDV-3（火鸡疱疹病毒1型，火鸡疱疹病毒，HVT）。MDV-1是诱导马立克氏病（MD）的唯一血清型，这是一种导致侵袭性T细胞淋巴瘤和麻痹症状的淋巴增生性疾病。在淋巴瘤和淋巴母细胞样细胞系（LCL）从他们衍生，MDV建立潜伏感染有限的病毒基因表达。LCL中的潜伏病毒基因组可以通过与鸡胚成纤维细胞（CEF）单层共培养来激活。MSB-1是从脾淋巴瘤中建立的第一个MDV转化的LCL之一，在携带致癌MDV-1和非致癌MDV-2病毒方面是不同的。在成功应用CRISPR/Cas9编辑方法精确敲除LCL中的MDV-1基因后，我们在这里描述了MS B-1细胞中MDV-2糖蛋白B（gB）的靶向缺失。由于gB对于感染性的本质，在MDV-2-gB缺失的MSB-1细胞中，CEF上MDV-2噬斑的产生被完全消除。我们的研究表明，CRISPR/Cas9系统可用于靶向灭活共感染的MDV-2，而不影响MSB-1细胞系中的MDV-1。本文证明的MDV-2的成功灭活也指出了使用靶向基因编辑作为抗病原性MDV-1和其他感染鸡的病毒的抗病毒策略的可能性。重要性马立克氏病（MD）是一种鸡的淋巴组织增生性疾病，其特征是多个器官中的快速发作的淋巴瘤和浸润到周围神经，导致瘫痪。来源于MD淋巴瘤的淋巴母细胞系（LCL）已成为提高对转化细胞中病毒-宿主相互作用（包括转化、潜伏和再活化）不同方面的理解的宝贵资源。MDV转化的LCL MSB-1来源于MDV BC-1株诱导的脾淋巴瘤，具有携带额外的非致病性MDV-2株HPRS-24的独特特征。通过在MS B-1细胞内从MDV-2基因组中靶向缺失必需基因糖蛋白B，我们证明了MDV-2病毒在共培养的CEF上的复制的完全抑制，而对MDV-1复制没有影响。鉴定的病毒基因对病毒的再激活/抑制至关重要，将可用作鸡对禽类病原体的从头疾病抗性的发展的靶点。
Marek’s disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are three different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek’s disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression. The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harboring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line. Successful inactivation of MDV-2 demonstrated here also points toward the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens. IMPORTANCE Marek’s disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency, and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harboring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.
影响因子：
3.7
作者：
Chi XJ;Lu YX;Zhao P;Li CG;Wang XJ;Wang M
通讯作者：
Wang M