Down-regulation of transcription of the proapoptotic gene BNip3 in cultured astrocytes by murine coronavirus infection

Abstract

Murine coronavirus mouse hepatitis virus (MHV) causes encephalitis and demyelination in the central nervous system of susceptible rodents. Astrocytes are the major target for MHV persistence. However, the mechanisms by which astrocytes survive MHV infection and permit viral persistence are not known. Here we performed DNA microarray analysis on differential gene expression in astrocyte DBT cells by MHV infection and found that the mRNA of the proapoptotic gene BNip3 was significantly decreased following MHV infection. This finding was further confirmed by quantitative reverse transcription-polymerase chain reaction, Western blot analysis, and BNip3-promoter-luciferase reporter system. Interestingly, infection with live and ultraviolet light-inactivated viruses equally repressed BNip3 expression, indicating that the down-regulation of BNip3 expression does not require virus replication and is mediated during cell entry. Furthermore, treatment of cells with chloroquine, which blocks the acidification of endosomes, significantly inhibited the repression of the BNip3 promoter activity induced by the acidic pH-dependent MHV mutant OBLV60, which enters cells via endocytosis, indicating that the down-regulation of BNip3 expression is mediated by fusion between viral envelope and cell membranes during entry. Deletion analysis showed that the sequence between nucleotides 262 and 550 of the 588-base-pair BNip3 promoter is necessary and sufficient for driving the BNip3 expression and that it contains signals that are responsible for MHV-induced down-regulation of BNip3 expression in DBT cells. These results may provide insights into the mechanisms by which MHV evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes.

Gene Expression Profiles of HIV-1-Infected Glia and Brain: Toward Better Understanding of the Role of Astrocytes in HIV-1-Associated Neurocognitive Disorders

“…Some of the genes induced in astrocytes by TNF-α or IL-1β described in this work were up-regulated by HIV-1 or SIV in vivo (Table 1). Other microarray analyses have shown that exposure of astrocytes in culture to different viruses (Cai et al 2003; Radhakrishnan et al 2003; Aravalli et al 2006; Rubio and Sanz-Rodriguez 2007), conditions of hypoxia (Mense et al 2006), and environmental toxins (Everall et al 2005; Mense et al 2006; Sengupta et al 2007) can all significantly alter gene expression profiles of these cells. While the results of such studies cannot be directly extrapolated to pathogenic situations in HIV-1-infected brains, they clearly indicate that astrocytes are highly reactive cells and that their physiological functions can be profoundly altered by contact with a wide variety of natural and pathogenic stimuli, including HIV-1.…”

Induction of transcription factor Egr-1 gene expression in astrocytoma cells by Murine coronavirus infection

“…First, we determined the BNip3 promoter activity by using the luciferase reporter assay . We expressed Egr-1 protein transiently in DBT cells by cotransfecting pBS-CMV-Egr-1, which contains the Egr-1 ORF under the control of a CMV promoter, and pGL3-BNip3 reporter plasmid (Bruick, 2000;Cai et al, 2003). After 20 h post-transfection, cell lysates were harvested and luciferase activity determined.…”

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

“…However, the mechanism of establishment of coronavirus persistence has not been yet well understood. Recently, the down-regulation of the pro-apoptotic protein BNip-3 was found in MHV-infected DBT cells by DNA microarray methodologies [2]. In MHV-infected cells, BNip3 levels were significantly decreased at the transcriptional level, and this down-regulation was mediated by the fusion between the viral envelope and the cell membrane.…”