Journal of Virology ( IF 5.4 ) Pub Date : 2017-10-01 , DOI: 10.1128/jvi.00992-17 Arundhati Gupta 1 , Darby G. Oldenburg 2 , Eduardo Salinas 1 , Douglas W. White 2 , J. Craig Forrest 1
Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo. Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo. kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo. These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68.
IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.
中文翻译:
表达卡波西肉瘤相关疱疹病毒潜伏期相关核抗原(LANA)的鼠γ疱疹病毒68揭示了LANA同系物的功能保守性和发散性。
潜伏期相关的核抗原(LANA)是由γ疱疹病毒的鼠李糖病毒属成员编码的多功能蛋白。使用鼠伽马疱疹病毒68(MHV68)进行的研究表明,LANA对于急性复制,潜伏期建立和体内再激活很重要。尽管在其DNA结合域(DBD)的结构上相似,但来自卡波西氏肉瘤相关疱疹病毒(KSHV)和MHV68的LANA同源物仍表现出相当大的序列差异。我们试图确定KSHV和MHV68 LANA同源物在功能上是否可互换。我们生成了编码KSHV LANA(kLANA)而不是MHV68 LANA(mLANA)的MHV68病毒,并评估了该病毒复制,建立和维持潜伏期以及重新激活的能力。kLANA敲入(KLKI)MHV68具有体外和体内复制能力,但与野生型(WT)MHV68相比,生长动力学更慢,滴度更低。接种小鼠后,KLKI MHV68在脾细胞和腹膜细胞中建立并维持了潜伏期,但没有有效地离体激活。kLANA抑制了ORF50的MHV68启动子,ORF50是编码主要裂解反式激活蛋白RTA的基因,而mLANA则不,这提示了KLKI MHV68表型的可能机制。通过提供MHV68 RTA在组织培养中反救KLKI MHV68复制来绕过这种抑制,并能够离体检测KLKI MHV68的活化。这些数据表明,kLANA和mLANA在功能上可互换,可用于建立和维持潜伏期,并表明kLANA对裂解复制的抑制(如先前与KSHV所示)是kLANA特有的功能,可转移至MHV68。
重要事项卡波西氏肉瘤相关疱疹病毒(KSHV)和鼠γ疱疹病毒68(MHV68)是γ疱疹病毒的Rhadinovirus属成员。这些病毒会建立终生感染,使它们各自的人类和鼠类宿主处于患癌症的危险中。潜伏期相关核抗原(LANA)是保守的鼠李糖病毒这些病毒长期长期感染所必需的蛋白质。为了更好地了解LANA同源物所执行的保守功能,我们生成了编码MHV68 LANA同源物的KSHV LANA蛋白的重组MHV68病毒。我们确定KSHV LANA蛋白能够在慢性感染的小鼠模型中支持MHV68潜伏期,而且还具有抑制病毒复制的功能。这项工作描述了一种体内模型系统,用于定义在鼠李糖病毒感染和疾病中LANA同源物在进化上保守和发散的功能。