Date of Award

Spring 1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Ann E. Campbell

Committee Member

Richard M. Stenberg

Committee Member

Timothy J. Bos

Committee Member

Richard P. Ciavarra

Committee Member

Robert E. Ratzlaff

Abstract

The purpose of this study was to identified nonessential murine cytomegalovirus (MCMV) genes involved in pathogenesis in vivo. Our approach to identifyjng these genes consisted of constructing MCMV mutants, and then analyzing these mutants in vitro and in vivo. Recombinant viruses (RV) expressing the β-glucuronidase marker gene were constructed by site-directed insertion and deletion mutagenesis of the MCMV Hind III-J and -I regions of the viral genome. Mutations were targeted to this region of the MCMV genome because the corresponding region of the human CMV genome is nonessential and is involved in down-regulating major histocompatibility complex (MHC) class I expression in infected human cells. Four mutant viruses were created: RV5, an insertion mutant in Hind III-J; RV6, a deletion mutant missing 2.8 kilobase (kb) pairs from Hind III-J; RV7, a deletion mutant lacking 7.7 kb from Hind III-J and -I; and RV9, a deletion mutant missing 10.7 kb from Hind III-J and -I. RV9 is a helper-dependent virus, and therefore, was not included in subsequent experiments. In vitro, RV5, RV6, and RV7 grew similarly to wild-type (WT) MCMV in NIH3T3 fibroblasts and in primary embryo fibroblasts, confirming that the mutations introduced into these viruses are nonessential for MCMV replication in cultured fibroblasts. In IC-21 macrophages, RV5 and RV6 great similarly to WT virus, however, RV7 grew 2 to 3 logs lower. A one-step growth curve of RV7 in these macrophages indicated that the reduced growth of this mutant virus was primarily due to a defect in virus replication. Northern blot analyses of immediate-early (IE), early, and late RNAs demonstrated that the block in RV7 replication occured at the IE phase of virus replication. A simultaneous Southern blot analysis of RV7-infected IC-21 macrophages indicated that RV7 entered these cells as efficiently as WT MCMV. Therefore, poor replication of RV7 in these cells is not the result of a defect in virus entry, but instead is due to inadequate IE viral gene expression. These data are highly significant because interactions between CMV and macrophages play a critical role in pathogenesis in vivo, particularly in virus dissemination and latency. The MCMV mutants were analyzed bv Western blot with an antibody to the H-2Kb class I heavy chain to determine if sequences within Hind III-J or -I were involved in the down-regulation of MHC class I expression observed in WT-infected cells. RV5, RV6, and RV7 significantly reduced the level of the class I heavy chain in infected fibroblasts, indicating that this nonessential region of the MCMV genome is not involved in class I down-regulation. In infected mice, all of the MCMV mutants displayed significantly reduced growth in the salivary glands, an organ central to the biology of CMV. In fact, the growth of RV7 in the salivary glands was barely detectable, indicating that the genes deleted from these viruses are required for the efficient replication of MCMV in the salivary gland.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/f3vg-am17

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