| Human cytomegalovirus (HCMV) is a member of the herpesvirus family, which includes herpes simplex virus1(HSV-1) and Epstein-Barr virus (EBV). This virus is the leading viral cause of abnormal development, sensorineural deafness and mental retardation in newborns. HCMV is a very important and common medical viruse that can infect almost anyone. During lytic infection, the proceeding of viral DNA synthesis, which occurs in the nucleus of infected cells, is one of the central events in viral life cycle. It's also a highly complex and regulated process that involves many internal protein-protein interactions within the replication-relative proteins and interactions with DNA replication initiation site, as well as interactions with the host celluar factors.This study is based on a previous systematic yeast two-hybrid library screens for eight HCMV DNA replication proteins by our lab. Using a yeast two-hybrid, co-immunoprecipitation assays and co-localization, the interactions of HCMV primase-associated factor (UL102) with a cellular transcriptional co-repressor C-terminal binding protein2(CtBP2) and the helicase (UL105) with Snap in were confirmed. The biological effects of these interactions then were further explored.The main function of HCMV primase-associated factor UL102according to previous report is to participate and modulate the activity of the helicase-primase complex (UL105, UL70and UL102). By either silencing or overexpressing the CtBP2protein and a modified oriLyt-dependent replication assay, we demonstrate for the first time that CtBP2acts as a restriction factor for Human Cytomegalovirus (HCMV) replication through inhibiting MIE gene expression and a direct interaction with UL102. Besides, an antagonistic relationship is found from this interaction, since representative HCMV late promoters can also be repressed by CtBP2but rescued by UL102indicated by dual Luciferase reporter experiments. Finally, CtBP2stable overexpression contributes to HCMV persistent infection at low MOI. HCMV UL105is believed to encode the helicase of the DNA replication machinery that tracks along the lagging strand and unwinds DNA in front of the replication fork. As an essential replication protein, UL105needs to localize in the nuclei, the site of viral DNA synthesis. In this study, we show that UL105specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and associated with cellular vesicles. The nuclear and cytoplasmic levels of UL105were decreased and increased in cells overexpressing Snapin, respectively, while the levels of UL105in the nuclei and cytoplasm were increased and decreased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Our results suggest that Snapin interacts with UL105and alters its cellular distribution, leading to modulation of viral DNA synthesis and progeny production.All the six HCMV-encoded core replication proteins (UL44, UL54, UL57, UL70, UL102and UL105) are highly conserved across all herpesviruses based on sequence similarities and biochemical properties and thus are the target for most of the current FDA-approved anti-herpes therapeutic agents. The research here facilitated the elucidations of anti-virus infection strategies of host cells against HCMV genome replication, the understandings of the antagonistic relationship between virus and host cells, and supplied the reference for the development of HCMV treatment project. |
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