The Regulatory Roles Of US28 Signal In Major Immediate Early Gene Induced Human Cytomegalovirus Activation

Human cytomegalovirus (HCMV) is a ubiquitous opportunisticβ-herpes virus that always causes congenital acute, latent and chronic infections. Although the primary infection is asymptomatic in immunocompetent individuals, the reactivation of the latent virus that follows from decreased immuno-surveillance can lead to a wide variety of diseases in immunocompromised hosts. Disease may involve the lung, liver, or nervous system. Death, sensorineural hearing loss, or mental retardation often results from HCMV infection of the fetus. HCMV infection has been implicated as an etiologic factor in arterosclerosis, immunologic rejection of allografts, or malignant tumor. The activation and replication of cytomegalovirus, which represents the most important event signaling the onset of virus-induced disease relapse, became the focus of HCMV-related disease research. The mechanism underlying maintenance of the latent viral genome and the switch between the latent and lyitc forms of HCMV infection remains unclear. Increasingly, research is focusing on methods to inhibit virus activation and prevent disease relapse.The major immediate early (MIE) gene of cytomegalovirus plays a key role in determining the activation of cytomegalovirus. It plays a critical role in the control of early and late viral gene expression, which is essential for viral replication. The abnormal expression of MIE is the marker of the HCMV activation. So, the enhancer upstream of the MIE gene and the MIE promoter are thought to influence the efficiency of transcription during virus lytic infection and viral reactivation. The factors that regulated the MIE transcription are viewed as important as MIE expression in the activation. Cellular infection by HCMV is associated with very early transcription and up-regulated expression of US28. The viral-encoded chemokine receptor homolog US28 changes occur even before the activation of the immediate-early virus genes. Interestingly, US28 has been shown to activate some transcription factors. Indeed, four binding sites for cAMP response element binding protein (CREB) have been found in the enhancer of MIE gene. However, the effect of US28 on MIE transcriptional activity through CREB remained unclear.So, whether US28 can constitutively activate CREB transcription factors, which can bind to the promoter/enhancer of the MIE gene and activate its transcription, and whether the enhancement of MIE promoter/enhancer activity was related with HCMV activation still need more investigation.To elucidate the possible role of US28 signal in the pathogenesis of HCMV activation, we cloned the US28 gene from DNA of HCMV-infected fibroblast at 72 h post infection and constructed the eukaryotic expression vector carrying full length of US28 gene. The recombinant plasmid was selected and identified by PCR analysis and DNA sequencing.To indicate the downstream effect of US28 on the active form of CREB phospho-CREB(p-CREB), HEK293 cells were transfected with the recombinant plasmid, and p-CREB was determined by western blot. We reported that cytomegalovirus-edcoded receptor US28 could enhance the p-CREB expression. Importantly, US28 could up-regulate p-CREB expression in a concentration-dependent manner in HEK293 cells. This result pointed out that US28 might enhance the transcription of CREB-related genes. And the forskolin, which is a direct activator of adenylate cyclase, could also up-regulate p-CREB expression in a concentration-dependent manner. But A-CREB (a dominant-negative inhibitor of CREB) did not effect p-CREB expression.To examine the role of CREB in MIE transcription induced by US28, the US28 recombinant plasmid and A-CREB were cotransfected with reporter plasmids (MIEP-Luc or pCRE-Luc) along with the pRL-TK vector, which is a control vector. The MIEP-Luc was a reporter plasmid which contained luciferase gene droved by the HCMV MIE enhancer/promoter. The pCRE-Luc was a luciferase reporter gene which had a promoter with three repeats of the CRE element within the sequences from–168 to +45 of theα-subunit of the human glycoprotein hormone gene. Using reporter gene assays in HEK293 cells, we found that US28 enhanced the transcriptional efficiency of MIE gene. This induction also activated theα-subunit of the human glycoprotein hormone gene promoter, which is sensitive to CREB. Then the reporter gene assays showed that, in the presence of US28, the enhanced MIE transcription effect could be partially blocked by A-CREB or enhanced by forskolin. So, there was a direct correlation among US28, CREB and transcription of MIE gene. US28 activated the MIE promoter/enhancer via CREB. The results showed that US28-induced the activity of MIE promoter/enhancer is partly regulated by CREB and that the involvement of other signal transduction routes and cellular factors independent of CREB.To detecte the relationship between the MIE expression and HCMV activation, we established real-time fluorescence polymerase chain reaction for HCMV copies and MIE expression detection in the process of HCMV activation. The quantitative real-time PCR assay was sensitive and specific, which showed that MIE expression began at the very early of the HCMV infection and was in a time-dependent manner in human embryo lung fibroblasts (HEL) cells. The copies of HCMV showed sharp enhancement behind the expression of MIE.We concluded that US28 enhanced MIE promoter/enhancer activity, and CREB was one of the multiple transcription factors involved in this process. The activation of the MIE gene was a key event in the cytomegalovirus replication cycle and was dependent on cellular transcription factors activated by viral US28. Besides, CREB is a transcription activator linked to a broad range of cellular processes. This indicated that US28 may affect the expression of many other genes, which are related to viral replication and dissemination. Based on the constitutive signaling of US28, it seemed likely that it may function in the reprogramming of the cell during HCMV infection.These results may have implications in the treatment of life-threatening systemic infections in immunocompromised patients. Consequently, drugs that target US28 may be efficient antiviral drugs. On the other hand, we might exploit the reconstructed US28 molecular to constitutively activate some target genes. The results provided the preclinical frameworks for novel therapeutics targeting at US28 to improve the patient outcome in HCMV disease. The real-time fluorescence polymerase chain reaction for HCMV copies and MIE expression detection laid the foundation for further study on screening and verifying latent infected cells, evaluating the therapeutic efficacy of antiretroviral treatment.