Activity-dependent changes in gene expression involving the transcription factor cAMP-response element-binding protein (CREB) occur in learning and memory, pain, and drug addiction. This mechanism may also be important for cytomegaloviral infections of the brain. The human cytomegalovirus major immediateearly promoter/enhancer (hCMV promoter), rate-limiting for productive cytomegalovirus infection, contains five cAMP-response elements (CREs). Indirect evidence suggests that this promoter does not function in unstimulated neurons. Here we test the hypothesis that expression from the hCMV promoter in neurons is induced by membrane depolarization. For these experiments, we infected cultured sympathetic and hippocampal neurons with hCMV-green fluorescent protein (GFP) promoter/reporter constructs using adenoviral gene transfer techniques and measured transgene expression by quantifying GFP fluorescence and GFP mRNA levels. We found that depolarization up-regulates promoter activity by >90-fold. Moreover, our results from pharmacological experiments suggest that this induction occurred through a CREB-dependent pathway. Importantly, site-directed mutagenesis of all five CREs in the promoter blocked this up-regulation almost completely, whereas mutating four of them had no effect. We conclude that the hCMV promoter acts as a molecular switch in neurons and is strongly induced by membrane depolarization, neuronal activity, or other stimuli that activate CREB. These results may provide insight into molecular mechanisms of cytomegalovirus-related diseases of the brain.
Human cytomegalovirus (hCMV)1 infections, often harmless in healthy individuals, produce numerous debilitating symptoms in those with immature or impaired immune systems. In persons with AIDS, for example, cytomegalovirus infections cause severe neurological disorders (1); in infants, congenital infections cause impaired brain growth, mental retardation, and progressive motor deficits (2). Therefore, it is important to understand the mechanisms for cytomegalovirus production in the nervous system. Productive cytomegaloviral infections require transcription of immediate-early genes in the viral genome (3, 4). Expression of these genes is controlled by the hCMV major immediate-early promoter/enhancer (hCMV promoter) (5). As activity of this promoter is rate-limiting for productive viral infection, many groups have studied how this promoter functions to understand the molecular mechanisms of cytomegalovirus infections. The hCMV promoter is highly enriched with a densely packed array of cis-elements (3) and is among the strongest known promoters in mammalian cells. As such, it is commonly used to drive the expression of foreign genes in a wide variety of cell types. Surprisingly, the hCMV promoter appears not to function well in neurons. For example, studies using primary cortical cultures indicate that the hCMV promoter expresses transgenes well in glia but not in neurons (6). Furthermore, in mice transgenic for hCMV promoter-reporter constructs, only a few ne...