RASCAL: a new tropism determinant encoded by human cytomegalovirus?

描述(由申请人提供)：人类巨细胞病毒(CMV)异常广泛的组织嗜性是该病毒在免疫受损个体中引起的严重多器官疾病的重要因素。除了淋巴细胞和多形性核白细胞外，几乎所有类型的细胞都能在体内支持病毒复制。尽管这种病毒在发病机制的发展中具有广泛的趋向性，但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特性和功能仍不清楚。本研究的目的是确定本课题组最近发现的一种新的病毒蛋白是否是CMV嗜性上皮细胞(EPC)、内皮细胞(EC)和树突状细胞(DC)的介体，这三种细胞在体内感染过程中对病毒进入、传播和免疫逃避起着基础性作用。为了确定可能参与介导特定细胞类型的病毒感染的新的病毒蛋白，比较了广亲和株TB40/E与高度趋向性的Towne株的编码内容。根据Towne编码的较长的异构体可能是无功能的假设，选择了一个推测的开放阅读框架(ORF)，该框架预测在TB40/E和Towne编码的蛋白中分别编码97aa和176aa的蛋白质。针对预测的氨基酸序列第21-39位氨基酸的多克隆抗体表明，该ORF确实在感染期间表达，并且编码的蛋白在感染后的晚期定位于核缘。基于这种特殊的亚细胞定位，我们将这个基因产物命名为核环相关巨细胞病毒蛋白，或Rascal。RASCALTB40/E和RASCALTowne在细胞内的定位没有差异，在病毒粒子从细胞核中排出的过程中，这两种亚型仍然与Lamin B阳性的细胞质小泡有关。最近完成的对纯化的病毒粒子的免疫印迹分析进一步表明，这两种蛋白可能都包含在成熟的病毒颗粒中。因此，我们推测，作为病毒粒子的组成部分，Rascal可能通过促进穿透的病毒粒子向细胞核的运输或在进入过程中病毒基因组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。我们还认为，这些功能的实现可能会因为Rascal亚型中额外的79个氨基酸的存在而受到阻碍，从而限制了编码它的病毒株的趋向性范围。在这项提案中，我们试图确定Rascal是否是一种被膜蛋白，以及它是否在EPC、EC和DC的病毒趋向性中发挥作用。 公共卫生相关性：巨细胞病毒(CMV)感染是免疫受损个体(如艾滋病患者、移植受者和新生儿)发病率和死亡率的严重来源。因此，开发新的策略来阻断病毒感染和免疫抑制是当务之急。我们的分析将确定新发现的CMV编码的蛋白质是否参与介导上皮细胞、内皮细胞和树突状细胞的病毒感染，这三种细胞类型与病毒在体内的增殖、传播和免疫逃避高度相关。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的抑制剂提供一个新的目标。

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.