RASCAL: a new tropism determinant encoded by human cytomegalovirus?

描述（由申请方提供）：人巨细胞病毒（CMV）异常广泛的组织嗜性是该病毒在免疫受损个体中引起严重多器官疾病的重要原因。几乎所有的细胞类型，除了淋巴细胞和多形核白细胞，可以支持病毒在体内复制。尽管这种病毒的广泛嗜性在发病机制的发展中的重要性，介导病毒进入各种不同细胞类型的分子机制，以及所涉及的蛋白质的身份和功能仍然不清楚。本提案的目标是确定我们小组最近鉴定的一种新的病毒蛋白是否是CMV对上皮细胞（EPC）、内皮细胞（EC）和树突细胞（DC）的嗜性的介体，这三种细胞类型在体内感染期间在病毒进入、传播和免疫逃避中发挥重要作用。为了鉴定可能参与介导特定细胞类型的病毒感染的新病毒蛋白，将广泛嗜性菌株TB 40/E的编码内容与具有高度有限嗜性的Towne菌株的编码内容进行比较。基于Towne编码的较长同种型可能无功能的假设，选择了一个推定的开放阅读框（ORF）进行进一步分析，该ORF预测在TB 40/E中编码97 aa的蛋白质，在Towne中编码176 aa的蛋白质。针对预测氨基酸序列的残基21-39的多克隆抗体显示，该ORF确实在感染期间表达，并且编码的蛋白质在感染后的后期定位于核边缘。基于这种特定的亚细胞定位，我们将这种基因产物命名为核环相关巨细胞病毒AL蛋白，或RASCAL。在RASCALTB 40/E和RASCALTowne的细胞内定位中没有观察到差异，并且两种亚型在病毒体从细胞核排出期间仍然与核纤层蛋白B阳性胞质囊泡相关。最近完成的纯化病毒粒子的免疫印迹分析进一步揭示，这两种蛋白质可能包含在成熟的病毒颗粒。因此，我们推测，作为一种病毒体组分，RASCAL可能通过增强穿透的病毒体向细胞核的运输或病毒基因组在进入过程中的核沉积来促进特定细胞类型的感染，这是在EPC、EC和DC中建立生产性感染的两个关键步骤。我们还建议，这些功能的实施可能会受到阻碍的存在下，额外的79个氨基酸在较长的RASCAL亚型，从而限制了编码it. In此建议的病毒株的嗜性范围，我们试图确定如果RASCAL是一个被膜蛋白，如果它发挥了作用，在病毒嗜性EPC，EC和DC。 公共卫生相关性：巨细胞病毒（CMV）感染是免疫受损个体如AIDS患者、移植受者和新生儿的发病率和死亡率的严重来源。因此，开发阻断病毒感染和免疫抑制的新策略是当务之急。我们的分析将建立一个新发现的蛋白编码的CMV参与介导病毒感染的上皮细胞，内皮细胞和树突细胞，三个高度相关的细胞类型的病毒增殖，传播和免疫逃避在体内。如果成功，这项研究将为设计改进的病毒传播和感染相关组织损伤抑制剂提供新的靶点。

DESCRIPTION (provided by applicant): The exceptionally broad tissue tropism of human cytomegalovirus (CMV) is a substantial contributor to the severe multi-organ disease caused by this virus in immune compromised individuals. Virtually all cell types, with the exception of lymphocytes and polymorph nuclear leukocytes, can support viral replication in vivo. Despite the importance of this virus' wide tropism in the development of pathogenesis, the molecular mechanisms mediating viral entry into a variety of different cell types, and the identity and function of the proteins involved are still poorly defined. The goal of this proposal is to determine whether a new viral protein recently identified by our group is a mediator of CMV tropism for epithelial (EPC), endothelial (EC) and dendrite cells (DC), three cell types that play fundamental roles in virus entry, dissemination and immunoevasion during infection in vivo. To identify new viral proteins potentially involved in mediating virus infection of specific cell types, the coding content of the broadly tropic strain TB40/E was compared to that of Towne, a strain with highly limited tropism. A putative open-reading frame (ORF) predicted to encode a protein of 97 aa in TB40/E and of 176 aa in Towne was selected for further analyses, based on the hypothesis that the longer isoform encoded by Towne might be non-functional. Polyclonal antibodies raised against residues 21-39 of the predicted amino acids sequence revealed that this ORF was indeed expressed during infection, and that the encoded protein localized to the nuclear rim at late times post-infection. Based on this specific sub cellular localization, we named this gene product nuclear Rim Associated CytomegalovirAL protein, or RASCAL. No difference was observed in the intracellular localization of RASCALTB40/E and RASCALTowne, and both isoforms remained associated with lamin B-positive cytoplasmic vesicles during virion egress from the nucleus. Recently completed immunoblot analyses of purified virions further revealed that both proteins might be contained in mature viral particles. We thus speculate that, as a virion component, RASCAL might promote infection of specific cell types by enhancing trafficking of penetrated virions towards the nucleus, or the nuclear deposition of the viral genome during entry, two critical steps for the establishment of productive infections in EPC, EC and DC. We also suggest that implementation of these functions might be hampered by the presence of the additional 79 amino acids in the longer RASCAL isoform, thus limiting the tropism range of viral strains encoding it. In this proposal we seek to determine if RASCAL is a tegument protein, and if it plays a role in viral tropism for EPC, EC and DC. PUBLIC HEALTH RELEVANCE: Cytomegalovirus (CMV) infections are a serious source of morbidity and mortality for immune compromised individuals such as AIDS patients, transplant recipients, and newborns. Because of this, the development of novel strategies to block viral infection and immune-suppression is a priority. Our analyses will establish if a newly identified protein encoded by CMV is involved in mediating viral infection of epithelial, endothelial and dendrite cells, three highly relevant cell types for viral proliferation, dissemination and immunoevasion in vivo. If successful, this research will provide a new target for the design of improved inhibitors of viral dissemination and infection-associated tissue damage.