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HCMV在培养细胞中溶解感染的基本分子生物学方面已经取得了很大的进展，尽管还有很多东西需要学习，但许多研究HCMV溶解感染的工具是可用的。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，尤其是小鼠巨细胞病毒，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。监管要素并不总是保守的。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，恒河巨细胞病毒（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性，有一些显著的差异。即使在小动物模型中编码调控蛋白的基因差异很大，在RhCMV中也相对保守。最重要的是，恒河猴的感染似乎在许多重要细节上概括了人类宿主的HCMV感染。因此，RhCMV被假设为研究CMV发病机制中无法在小动物模型中解决的那些方面的理想模型。不幸的是，证实这一假设和有效利用这一系统所需的基本分子信息还没有得到。具体目标是：1)建立包含整个RhCMV基因组的有序的一组或多组cosmid克隆，2)确定和分析RhCMV基因组的完整核苷酸序列，以及3)开发生成重组的系统，从而能够有效地构建突变病毒，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其他棘手问题。

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.