Genetic engineering of murine cytomegalovirus: Mapping essential and non-essential genes.

摘要

Human cytomegalovirus (HCMV) causes mild or subclinical diseases in immunocompetent adults but may lead to severe complications in immunosuppressed, immunocompromised, or immature immune function individuals. In order to fully understand CMV viral pathogenesis, it is essential to perform studies on the function of viral genes. The overall objective of this dissertation is to determine the role of murine cytomegalovirus (MCMV) genes in viral replication: classification of essential and non-essential genes through the creation and analysis of deletion mutant viruses. In addition, we further characterized non-essential mutant viruses based on their growth properties in NIH3T3 murine fibroblast cells. To achieve this objective, the development of different genetic techniques to generate viral mutants was undertaken. The first technique addressed was the generation of MCMV mutant viruses by replacing open reading frames with green fluorescent protein (GFP). Results from this section indicate m09 and M43 are not essential for replication in NIH3T3 cells. m09 is not essential for viral growth in spleen, lungs, liver, kidneys, and salivary glands of SCID mice. M43 is not essential for viral growth in the spleen, lungs, liver, and kidneys but is essential for growth in the salivary glands of SCID mice. Most importantly, deletion of the desired ORF was possible and viruses can be easily visualized with the GFP reporter protein. The next technique addressed was the protocol for generating bacterial artificial chromosome (BAC) mutants using a lab generated BAC, pMBAC3637. Results from this part demonstrated that mutants can be generated in a rapid and efficient way with the BAC. Concerns for the amount of labor and the cost of infecting large groups of mice lead us to the third technique. The generation of luciferase BAC mutants that allow us to visualize viral infection in the mouse in real time without sacrificing the animal. Finally, the advent of a full length BAC, pSM3fr, that replicates as well as the Smith strain virus both in vitro and in vivo combined with the generation of a BAC mutant protocol allowed mutagenesis of the complete MCMV genome. Every open reading frame of the MCMV Smith genome was deleted and phenotypic studies were carried out on the resulting mutant viruses.