Generation of a Reporter Cell Line for Detection of Infectious Varicella-Zoster Virus and Its Application to Antiviral Studies

Abstract

To simplify the titration of infectious varicella-zoster virus (VZV), we generated a reporter cell line that produced luciferase in a dose-dependent manner upon infection with cell-free VZV. A few VZV-infected cells were detectable by coculturing with the cell line. We demonstrated the usefulness of the cell line for antiviral studies.Varicella-zoster virus (VZV) causes varicella on primary infection and zoster upon reactivation. Both conditions may be associated with severe complications (1). For the treatment of VZVassociated diseases, acyclovir (ACV), valaciclovir, and famciclovir have been used widely (1,25). The number of approved drugs for treating VZV diseases is limited, and all of them have the same mode of action. The development of resistant strains and the ineffectiveness of available antivirals at preventing complications have necessitated a search for alternative compounds (7,8,25,33). Several new compounds have been under investigation (13,20,21,31,32). Plaque reduction assays have been routinely used to screen and evaluate candidate compounds, but this method is made laborious and time consuming due to the slow growth of VZV in tissue culture. Some methods aimed at reducing the inconvenience have been developed (26,27,34), as has a biochemical assay for characterization of ACV-resistant strains (29). However, the unavailability of a rapid, high-throughput assay for titrating VZV has hampered efforts both to screen new compounds and to detect drug-resistant strains in a timely fashion.Previously, we established a reporter cell line for human herpesvirus 8 and demonstrated its practical uses (10,11,14). We report here the generation of a similar cell line for VZV by use of the following approach: (i) selection of the most suitable promoter from various promoters, (ii) identification of the minimum sequence length for efficient activation of the selected promoter to avoid nonspecific activation, (iii) establishment of a cell line that contains the reporter gene under the control of the minimum promoter, and (iv) characterization of the clone that performed best.Characterization of individual VZV promoters and global VZV gene expression have been reported previously (4, 5, 12).On the basis of those studies, various DNA fragments containing known or putative VZV promoters (Table 1) were amplified from the nucleocapsid DNA of VZV pOka strain by PCR, cloned into a luciferase vector pGL3-Basic, and then analyzed for their responses to VZV infection in transient transfection experiments using BSC40 cells (3) and MeWo cells (9). Among the promoters examined, the ORF9 promoter was most strongly activated in both cell lines (Fig. 1). Activation of each promoter was also analyzed by cotransfection with a plasmid expressing VZV IE62. The correlation between infection-and IE62-mediated activation (Fig. 1C) was good, indicating that infection-mediated activation was VZV specific.The minimal essential region for the ORF9 promoter was

The Ubiquitous Cellular Transcriptional Factor USF Targets the Varicella-Zoster Virus Open Reading Frame 10 Promoter and Determines Virulence in Human Skin Xenografts in SCIDhu Mice In Vivo

“…Transient-transfection experiments documented that the ORF10 promoter was strongly responsive to the major VZV immediate-early transactivator, IE62, as has been observed with many other VZV genes (43). Recently studies of several VZV promoters showed that their activities induced by VZV infection didn't differ from that mediated by IE62 alone, suggesting that the synergistic effects on the promoter activations induced by VZV infection could be reflected by IE62-mediating activation (49). There was little or no response to other known VZV transactivators, including IE4, the ORF61 protein, and IE63.…”

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

“…Similar cell lines have been described for several other viruses (34). We and others generated reporter cell lines for herpesviruses, including herpes simplex virus, B virus, HHV-8, and VZV (7,17,34,42,47). Two reporter cell lines for HCMV that depend on the activation of the UL54 (DNA polymerase) promoter were also published previously (13,45).…”

“…MeWo cells were used for infection with VZV (P-Oka). MV9G, a VZV reporter cell line, was used for titration of VZV (47). A telomeraseimmortalized human fibroblast cell line, hTERT-BJ1 (Invitrogen), was grown in Dulbecco's modified Eagle medium-medium 199 (4:1) supplemented with 10% FBS and infected with the HCMV Towne and RC256 (41) strains and with several HCMV clinical isolates.…”

“…To reduce the inconvenience, several modified high-throughput assays, including the use of recombinant viruses expressing a marker gene product, colorimetric assays, and flow cytometric assays, have been described (1,5,13,27,28,39). Previously we established reporter cell lines for human herpesvirus 8 (HHV-8) and varicella-zoster virus (VZV) and demonstrated their practical uses (17,25,47). In this study, we generated a similar cell line for HCMV.…”

Identification of a Varicella-Zoster Virus Replication Inhibitor That Blocks Capsid Assembly by Interacting with the Floor Domain of the Major Capsid Protein

“…EC 50 s of 35B2 against several laboratory and clinical strains were determined by the focus reduction assay using cell-associated viruses as inocula, since most low-passage-number VZV strains are cell associated. It is well known that EC 50 s obtained by using cell-associated viruses as inocula are higher than those obtained by using cell-free viruses (44,57). Growth of ACV-resistant strains V-Oka-YSR and Kanno as well as other ACV-sensitive strains and clinical isolates was inhibited by the 35B2 treatment at similar concentrations ( Table 2).…”