Recombinant Green Fluorescent Protein-Expressing Human Cytomegalovirus as a Tool for Screening Antiviral Agents

Abstract

A recombinant human cytomegalovirus (AD169-GFP) expressing green fluorescent protein was generated by homologous recombination. Infection of human fibroblast cultures with AD169-GFP virus produced stable and readily detectable amounts of GFP signals which were quantitated by automated fluorometry. Hereby, high levels of sensitivity and reproducibility could be achieved, compared to those with the conventional plaque reduction assay. Antiviral activities were determined for four reference compounds as well as a set of putative novel cytomegalovirus inhibitors. The results obtained were exactly in line with the known characteristics of reference compounds and furthermore revealed distinct antiviral activities of novel in vitro inhibitors. The fluorometric data could be confirmed by GFP-based flow cytometry and fluorescence microscopy. In addition, laboratory virus variants derived from the recombinant AD169-GFP virus provided further possibilities for study of the characteristics of drug resistance. The GFP-based antiviral assay appeared to be very reliable for measuring virus-inhibitory effects in concentration-and time-dependent fashions and might also be adaptable for high-throughput screenings of cytomegalovirus-specific antiviral agents.Human cytomegalovirus (HCMV), a betaherpesvirus, is a major opportunistic pathogen of humans with a worldwide distribution. Primary infection with HCMV in persons with a normal immune system is generally asymptomatic, while in rare cases a self-limiting, mild mononucleosis syndrome, or even more severe manifestations, may develop. In contrast, in immunocompromised persons (e.g., organ transplant recipients), HCMV frequently causes systemic disease with typical clinical consequences, like retinitis, pneumonitis, or gastroenteritis. The incidence of HCMV disease in AIDS patients, in comparison, has dramatically decreased since the availability of potent antiretroviral therapy. Furthermore, congenital infection is a major problem with HCMV, since this may result in a severe, generalized cytomegalic inclusion disease (CID) of the neonate (reviewed in reference 6).At present, clinically available drugs for antiviral therapy include the inhibitors of viral DNA polymerase, ganciclovir (GCV; also called Cytovene or Cymeven), foscarnet (FOS; also called Foscavir), and cidofovir (CDV; also called Vistide). All these drugs have low oral bioavailability and dose-related toxicities (13, 27); consequently, novel antiviral compounds with improved efficacy and fewer side effects are needed. Currently, several drugs with anti-HCMV activity are in preclinical or clinical evaluations. These include a series of benzimidazole riboside compounds (BDCRB, TCRB, 1263W94, and others) showing efficient inhibition of various steps in HCMV replication (e.g., genomic DNA maturation) (18, 33; reviewed in reference 9). Another attractive inhibitor candidate was described by studies on the phosphorothioate oligonucleotide fomivirsen (ISIS 2922), which specifically binds to sequences complementary to the major...

The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import

“…To infect HFFs with recombinant HCMVs, cells were inoculated with appropriate multiplicities of viral stocks for 90-120 min at 37 uC, before virus inoculi were removed and replaced with fresh growth medium. Viral growth curves were determined in HFFs by the use of an established HCMV-GFP-based replication assay (Marschall et al, 2000). In brief, progeny-viruscontaining supernatants of infection kinetics were collected and transferred to freshly seeded HFFs in 12-well plates.…”

“…In brief, progeny-viruscontaining supernatants of infection kinetics were collected and transferred to freshly seeded HFFs in 12-well plates. Seven days postinfection, all wells with HCMV-infected cells were harvested, lysed and processed for automated GFP fluorometry as described previously (Marschall et al, 2000;Herget et al, 2004).…”

Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

“…Human primary foreskin fibroblasts (HFFs) were cultivated in minimal essential medium containing 7.5 % FCS. HCMV strains AD169, AD169-GFP (Marschall et al, 2000) and BAC213 (AD169delUL97-GFP; Marschall et al, 2005) were propagated in HFFs and used for infection assays as described previously (Marschall et al, 2000). Transfections were performed by using Lipofectamine 2000 (Invitrogen), polyethylenimine (Sigma; Schregel et al, 2007) or calcium phosphate-DNA complexes (Hofmann et al, 2000;Winkler et al, 1994).…”

Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase

“…S4) from an intergenic locus between US9 and US10, a site that has been used by others to express heterologous genes (30).…”