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功能低下个体中引起严重多器官疾病的重要因素。除了淋巴细胞和多态核白细胞外，几乎所有的细胞类型都能支持病毒在体内的复制。尽管这种病毒的广泛趋向性在发病机制的发展中具有重要意义，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身份和功能仍然不清楚。本提案的目的是确定我们小组最近发现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）趋向性的介质，这三种细胞类型在体内感染期间病毒进入、传播和免疫逃逸中起着重要作用。为了鉴定可能参与介导特定细胞类型病毒感染的新病毒蛋白，我们将广向性毒株TB40/E的编码内容与高度有限向性毒株Towne的编码内容进行了比较。基于Towne编码的较长同种异构体可能无功能的假设，我们选择了一个假定的开放阅读框架（ORF）进行进一步的分析，预计在TB40/E中编码一个97 aa的蛋白，在Towne中编码一个176 aa的蛋白。针对预测氨基酸序列残基21-39的多克隆抗体显示，该ORF确实在感染期间表达，并且编码蛋白在感染后后期定位于核边缘。基于这种特异的亚细胞定位，我们将该基因产物命名为核环相关巨细胞病毒蛋白（nuclear Rim Associated CytomegalovirAL protein， RASCAL）。RASCALTB40/E和RASCALTowne在细胞内的定位没有差异，在病毒粒子离开细胞核时，这两种异构体都与层粘连蛋白b阳性的细胞质囊泡有关。最近完成的纯化病毒粒子的免疫印迹分析进一步表明，这两种蛋白可能包含在成熟的病毒颗粒中。因此，我们推测，作为一种病毒粒子成分，RASCAL可能通过增强穿透病毒粒子向细胞核的运输或病毒基因组在进入细胞核时的核沉积来促进特定细胞类型的感染，这是在EPC、EC和DC中建立生产感染的两个关键步骤。我们还认为，这些功能的实现可能受到RASCAL亚型中额外的79个氨基酸的存在的阻碍，从而限制了编码它的病毒株的向性范围。在本研究中，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的病毒趋向性中起作用。

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.