Establishment of a Cell-Based Assay for Screening of Compounds Inhibiting Very Early Events in the Cytomegalovirus Replication Cycle and Characterization of a Compound Identified Using the Assay

Abstract

To simplify the detection of infectious human cytomegalovirus (HCMV), we generated a cell line that produced luciferase in a dose-dependent manner upon HCMV infection. Using this cell line, we identified anti-HCMV compounds from a diverse library of 9,600 compounds. One of them, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide (DPPC), was effective against HCMV (Towne strain) infection of human lung fibroblast cells at a 50% effective concentration of 2.5 M. DPPC also inhibited the growth of clinical HCMV isolates and guinea pig and mouse cytomegaloviruses. Experiments using various time frames for treatment of the cells with DPPC demonstrated that DPPC was effective during the first 24 h after HCMV infection. DPPC treatment decreased not only viral DNA replication but also IE1 and IE2 expression at mRNA and protein levels in the HCMV-infected cells. However, DPPC did not inhibit the attachment of HCMV particles to the cell surface. DPPC is a unique compound that targets the very early phase of cytomegalovirus infection, probably by disrupting a pathway that is important after viral entry but before immediate-early gene expression.

Mood stabilizers inhibit cytomegalovirus infection

“…Quantitative real time PCR (qPCR) assays for hCMV UL132 (Pa03453400_s1) and human albumin (Hs99999922_s1) genes were performed using TaqMan gene expression assays (Life Technologies) [40, 41]. Ten-fold dilutions of hCMV DNA and cellular DNA from human fibroblasts were used as quantitative standards.…”

A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line

“…In most cases, reporter genes controlled by HCMV promoters were inserted into the HCMV-susceptible human glioma cell line U373-MG [16–18] or in mink lung cells [19]. Either firefly luciferase [16,17] or green fluorescent protein (GFP) [18,19] have been chosen as reporters in these studies. Different HCMV early promoters were used to control reporter gene expression: pUL54 [17–19], pUL112/113 [18] or pTRL4 [16].…”

“…Either firefly luciferase [16,17] or green fluorescent protein (GFP) [18,19] have been chosen as reporters in these studies. Different HCMV early promoters were used to control reporter gene expression: pUL54 [17–19], pUL112/113 [18] or pTRL4 [16]. The promoters have in common that they are activated only by HCMV infection and not by infection with human alpha- or other betaherpesviruses (herpes simplex virus type 1 and 2 [17–19]; Varicella-zoster virus [16,19]; human herpesvirus type 6 [16]).…”

A dark-to-bright reporter cell for classical swine fever virus infection

“…The basic strategy is to stably introduce genetic elements into a cell in such a way that when a particular virus enters in the cell, a virus-specific event that results in the production of an easily measurable enzyme is triggered (Olivo, 1996;Leland and Ginocchio, 2007). Cell lines expressing reporter genes inducible upon viral infection have shown to be both sensitive and specific (Olivo, 1996;Lee et al, 2004;Lutz et al, 2005;Wang et al, 2006;Ueno et al, 2006;Wu et al, 2007;Fukui et al, 2008;Li et al, 2009;Iro et al, 2009;Hossain et al, 2010;Levy et al, 2010). These reporter cells exploit the specificity of viral infection in combination with the extreme sensitivity of reporter proteins such as luciferase, chloramphenicol acetyltransferase (CAT), LacZ (b-D-galactosidase), green fluorescence protein (GFP) or secreted alkaline phosphatase (SEAP).…”