Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.

Constant up-regulation of BiP/GRP78 expression prevents virus-induced apoptosis in BHK-21 cells with Japanese encephalitis virus persistent infection.

BACKGROUND: Persistent infection of the Japanese Encephalitis Virus (JEV) has been reported in clinical cases, experimental animals, and various cell culture systems. We previously reported the establishment of spontaneous JEV persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in BHK-21 cells devoid of virus-induced apoptosis, cBS6-2 and cBS6-3. However, cell-specific factors may play important roles in controlling JEV replication and have never been assessed for this specific phenomenon. Recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (UPR). RESULTS: To identify the host cell factors that affect JEV persistence, we investigated the expression of essential UPR factors in cBS6-2 and cBS6-3 cells. Of the selected UPR factors tested, the most noticeable deviations from those of the normal BHK-21 cells with JEV acute infection were as follows: the suppression of C/EBP homologous binding protein (CHOP) and the constant up-regulation of immunoglobulin binding protein (BiP) expression in cBS6-2 and cBS6-3 cells. In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely. Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death. Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions. CONCLUSIONS: BHK-21 cells with JEV persistent infection strive against virus-induced apoptosis through constant up-regulation of BiP expression, resulting in the complete depletion of CHOP even with apparent virus amplification in the cells. Accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of JEV persistent infection in mammalian cells.

Extended stability of cyclin D1 contributes to limited cell cycle arrest at G1-phase in BHK-21 cells with Japanese encephalitis virus persistent infection.

There is increasing evidence that many RNA viruses manipulate cell cycle control to achieve favorable cellular environments for their efficient replication during infection. Although virus-induced G0/G1 arrest often delays early apoptosis temporarily, a prolonged replication of the infected virus leads host cells to eventual death. In contrast, most mammalian cells with RNA virus persistent infection often escape cytolysis in the presence of productive viral replication. In this study, we demonstrated that the extended endurance of cyclin D1 was clearly associated with the suppression of glycogen synthase kinase-3ß (GSK-3ß) expression in BHK-21 cells that are persistently infected with Japanese encephalitis virus (JEV). The G0/G1 arrest of these cells turned much loose compared to the normal BHK-21 cells with JEV acute infection. After cycloheximide treatment, cyclin D1 in the persistently infected cells lasted several hours longer than those in acutely infected cells. Furthermore, both p21(Cip1) and p27(Kip1), positive regulators for cyclin D1 accumulation in the nucleus, were suppressed in their expression, which contrasts with those in JEV acute infection. Inhibition of the GSK-3ß by lithium chloride treatment rescued a significant number of cells from cytolysis in JEV acute infection, which coincided with the levels of cyclin D1 that escaped from proteolysis. Therefore, the limitation of G1/S arrest in the BHK-21 cells with JEV persistent infection is associated with the suppression of GSK-3ß expression, resulting in the extended duration of cyclin D1.

Reliability of non-culturable virus monitoring by PCR-based detection methods in environmental waters containing various concentrations of target RNA.

Owing to the lack of practical cell culture system for human noroviruses (HuNoV), various detection methods based on conventional reverse transcription-PCR (RT-PCR) and the quantitative real-time PCR have been major tools for monitoring environmental water safety. In this study, we showed that the proportion of water sample concentrates used for one-step RT-PCR significantly influences false-negative findings of the non-culturable viruses. In total, 59 archived samples of previously analyzed water concentrates were reexamined for HuNoV RNA by the one-step RT-PCR and semi-nested PCR. Using new aliquots for RNA extraction for every trial, up to 20 PCR trials were performed for each archive to determine whether the crosscheck results supported the previous determinations. We reconfirmed that 27.6% (8/29) of the samples were HuNoV-positive samples: 6.7% (1/15) from groundwater, 33.3% (3/9) from river water, and 80% (4/5) from treated sewage effluent (TSE). These results corresponded to the ratio of previously negative HuNoV samples now identified as positive (8/30): 6.7% (1/15) from groundwater, 20% (1/5) from river water, and 60% (6/10) from TSE. To elucidate the cause of these results, 16 different concentrations of murine norovirus (MNV) RNA (from 2×10(2) to 8×10(3) copies, divided into 10 tubes for each concentration) were subjected to one-step RT-PCR. The detection frequency and reproducibility decreased sharply when the number of MNV RNA copies fell below threshold levels. These observations suggest that the proportion of water concentrate used for PCR-based detection should be considered carefully when deciding viral presence in certain types of environmental water, particularly in regard with legal controls.