Dengue virus (DENV) of the Flaviviridae family is the most significant cause of human arboviral disease worldwide. While other members of the Flaviviridae family can cause persistent infections, there are few reports of persistent DENV infections in mammalian cells. A DENV-2 mutant that can establish persistent infection in a range of cultured cells was previously discovered, and the phenotype was attributed to a single amino acid substitution in the DENV-2 NS4B protein, T66A. However, prior studies failed to reveal the mechanism conferring the persistent phenotype. To further address this question, a comparative RNAseq transcriptomic analysis using RNA from HEK293T cells that were infected with DENV-2, persistently-infected, or stably expressing a wild-type (RepDV-GP2A) or NS4BT66A¬ mutant replicon (RepDV-GP2A-NS4BT66A) was performed. Bioinformatic analysis of the transcriptomic dataset revealed that the expression of a range of interferon-stimulated genes was increased in the persistently-infected cells compared to control and DENV-2 infected cells. Immunomicroscopy revealed that the distribution of STAT1 was altered between persistently-infected and DENV-2 infected cells, indicating an effect of the NS4BT66A¬ mutation on DENV-induced modulation of the host interferon response. Interestingly, genes involved in cholesterol biosynthesis (HMGCR, HMGCS1, IDI1, INSIG1, MSMO1, SQLE) were downregulated in the persistently-infected cells compared to the control cells, but upregulated during DENV-2 infection. Downregulation of target genes was validated by quantitative real-time PCR. The distribution of intracellular cholesterol was also examined and found to be altered upon persistent and wild-type DENV-2 infection. Attempts to pharmacologically clear persistently-infected cells of virus were unsuccessful; thus, the persistently-infecting virus genome was sequenced to identify mutations that arose and potentially conferred resistance to the anti-DENV drugs. This study provides further insight into the mechanism by which persistent infection with the mutated DENV-2 strain, v601-4BT66A, is maintained in vitro.
|Date of Award||25 Jan 2019|
- The University of Bristol
|Supervisor||Andrew D Davidson (Supervisor)|