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|Title:||Characterization of RL13 in Human Cytomegalovirus (HCMV) Replication|
|Abstract:||Human cytomegalovirus (HCMV) is a ubiquitous -herpesvirus that is present in 60 to 99 percent of adults. After infection, the virus remains in the latent state for the lifetime of the host, but can later reactivate to generate new infectious virus. Although healthy people with the virus are generally asymptomatic, among immune-compromised individuals, the virus has been found to cause life-threatening diseases, including encephalitis and pneumonia. The HCMV strains, including clinical strains, adapt to in vitro culture even during preparation of virus stock in order to replicate efficiently in vitro; as a result, findings based on research strains may have limited applicability. Specifically, clinical samples of the virus adapt to cell culture by mutations in the RL13 gene, as well as other loci, including the UL128 locus. Repair of the RL13 mutation in vitro inhibits wild-type HCMV replication; however, the role of RL13 in HCMV replication is currently unknown. Its presence in the virion envelope has suggested that it plays a role in modifying tropism or modulating cell signaling during virus entry. To answer the question of why RL13 is wild-type in primary isolates but mutated in culture-adapted viruses in vitro, in this study, we sought to characterize the role of RL13 in HCMV replication. In particular, we sought to determine the effect of wild-type versus mutant RL13 on HCMV replication. We developed lentiviral vectors containing wild-type or mutant RL13, which we then used to transduce MRC-5 fibroblast cells to use for analysis of viral DNA replication, virus production, and the infectivity of progeny virions produced. While we found that overall genome production levels were not substantially different from HCMV- infected MRC-5 cells expressing wild-type RL13 compared to those expressing mutant RL13, we found that wild-type RL13 reduced production of infectious progeny virions as well as viral entry into fibroblast cells. These findings suggest that RL13 rapidly mutates in vitro in order to produce progeny virions with maximal infectivity to better adapt to cell culture. This work has implications for expanding our understanding of the mechanisms by which HCMV replicates and for the production of research strains of HCMV that more fully model the virus found in vivo, potentially enhancing the applicability of research findings and the development of treatments and, ultimately, a vaccine.|
|Type of Material:||Princeton University Senior Theses|
|Appears in Collections:||Molecular Biology, 1954-2016|
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