A MOLECULAR BASIS FOR THE RHESUS CYTOMEGALOVIRUS MODEL

人类巨细胞病毒仍然是一个重大的健康问题。 在了解培养细胞中HCMV裂解感染的基础分子生物学方面已经取得了很大进展，尽管还有很多东西有待了解，但已有许多研究HCMV裂解感染的工具。 然而，在组织培养中，对潜伏期和发病机制的全面研究是困难的或不可能的，并且对HCMV生物学的这些方面了解较少。小动物模型，最值得注意的是小鼠CMV，已被开发用于研究发病机制和潜伏期，并已被证明是非常宝贵的。 然而，这些模型在几个方面与人类系统有很大的不同。 许多基因已经分化得难以辨认。 调控元件并不总是保守的。 发病机制各不相同。 由于这些原因，它们不足以解决一些问题。 相比之下，恒河猴CMV（RhCMV）与HCMV非常相似。 有限的序列数据表明，整个基因组与人类病毒大致共线，有一些显着的差异。 即使是在小动物模型中差异很大的调控蛋白编码基因在RhCMV中也相对保守。 最重要的是，恒河猴中的感染似乎在许多重要细节上重演了人类宿主的HCMV感染。因此，假设RhCMV是研究CMV发病机制的那些方面的理想模型，这些方面不能在小动物模型中解决。 不幸的是，确认这一假设和有效地利用这一系统所需的基本分子信息还没有。 具体目标是：1）建立含有整个RhCMV基因组的一组或多组有序粘粒克隆，2）确定和分析RhCMV基因组的完整核苷酸序列，和3）开发用于产生重组体的系统，所述重组体将能够有效构建突变病毒，然后可以在体内评估其生物学特性。 这些目标将使用标准的分子克隆和测序方法，以及已经应用于其他疱疹病毒构建突变体的方法来解决。 本文提出的工作的长期目标是建立知识库，使RhCMV系统能够有效地应用于CMV发病机制和潜伏期研究中的棘手问题。

Human cytomegalovirus remains a significant health problem. Great strides have been made in understanding the basic molecular biology of HCMV lytic infection of cells in culture and, although much is yet to be learned, many tools to study HCMV lytic infection are available. However, comprehensive studies of the latent phase and pathogenesis are difficult or impossible in tissue culture, and these aspects of HCMV biology are less well understood. Small animal models, most notably murine CMV, have been developed to study pathogenesis and latency, and have proven invaluable. These models are nevertheless significantly divergent from the human system in several respects. Many genes have diverged beyond ready recognition. Regulatory elements are not always conserved. Aspects of pathogenesis differ. For these reasons, they are not adequate to address some questions. In contrast, the rhesus CMV (RhCMV) is very similar to HCMV. The limited sequence data available show that the overall genome is roughly colinear with the human virus, with a few notable differences. Even genes encoding regulatory proteins that are quite divergent in small animal models are relatively well conserved in RhCMV. Most importantly, infection in rhesus macaques appears to recapitulate in many important details infection by HCMV of the human host. Therefore, RhCMV is hypothesized to be an ideal model for the study of those aspects of CMV pathogenesis that cannot be addressed in the small animal models. Unfortunately, the basic molecular information required to confirm this hypothesis and efficiently exploit this system is not yet available. The specific aims are to: 1) establish an ordered set or sets of cosmid clones containing the entire RhCMV genome, 2) determine and analyze the complete nucleotide sequence of the RhCMV genome, and 3) develop systems for generating recombinants that will enable efficient construction of mutant viruses whose biological properties can then be assessed in vivo. These aims will be addressed using standard molecular cloning and sequencing methods, and methods already applied to other herpesviruses to construct mutants. The long-term goal of the work proposed here is to build the knowledge base that will enable efficient application of the RhCMV system to otherwise intractable problems in studies of CMV pathogenesis and latency.