The relationship between penetration and uncoating of poliovirus in HeLa cells

177

114

. Recently, the inhibitory activity of WIN 51711 against a number of enterovirus and rhinovirus serotypes has been reported (10) with reduction in plaque formation and reduction in virus yield at concentrations below that which inhibits cell growth. In plaque reduction assays, concentrations of WIN 51711 in the range of 0.004 to 0.17 ,ug/ml caused a 50% reduction in plaque formation for the enteroviruses tested. Generally, the rhinoviruses tested in plaque reduction assays were less sensitive to WIN 51711 than were the enteroviruses. However, the majority of the 33 serotypes which were tested showed a 50% reduction in plaque formation at less than 1 ,ug of WIN 51711 per ml (2.9 ,uM 433, 1984).Preliminary data presented here indicate that WIN 51711 exerts its virus-specific activity through the inhibition of an early event in the replication of rhinovirus and poliovirus and that the molecular basis of this mechanism is the inhibition of the virion uncoating process. Drug-resistant poliovirus type 2 variants were selected by picking plaques from plates infected with poliovirus in the presence of 1.0 and 0.1 ,ug of WIN 51711 per ml. The viruses isolated from plaques that developed in the presence of drug were passaged three times in HeLa (Ohio) monolayer cultures in the presence of 1.0 and 0.1 ,ug/ml and were designated rCl-1.0y and rCl-0.1y, respectively. These resistant variant stocks were stored at -70°C after testing for sensitivity to WIN 51711. MATERIALS AND METHODS VirusesVirus titrations were determined by plaque assay on day-old confluent HeLa (Ohio) monolayer cultures in sixwell cluster plates (Costar, Cambridge, Mass.

Section: Discussionsupporting

confidence: 75%

Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug

Fox¹,

Otto²,

McKinlay³

1986

177

114

“…Picornaviruses (Echo 12, Polio 2, and RV 1-A) made photosensitive with neutral red to measure viral uncoating were capable of losing photosensitivity during the first 3 h after infection of HeLa cells in the presence of antiviral concentrations of MDL-860. This was interpreted as evidence that viral uncoating was not affected (3,4). MDL-860 appeared to inhibit some early event in replication, after uncoating, which is required for synthesis of a majority of viral RNA.…”

Section: Resultsmentioning

confidence: 99%

Antiviral activity and mechanism of action of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860)

Torney¹,

Dulworth²,

Steward³

1982

A nitrobenzene derivative, MDL-860, was found to inhibit plaque formation, cytopathic effect, or both in 11 of 12 picornaviruses at concentrations which did not affect cell growth. The A search in The Dow Chemical Company laboratories for agents active against the picornaviruses led to the identification of the broadly active 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860). This agent exhibited high in vitro activity against the picornaviruses but not against viruses of other classes. This report is concerned with the spectrum of activity of MDL-860 and the results of some preliminary observations on its mechanism of action. The accompanying report (5) presents a more extensive evaluation of the antiviral spectrum of the compound. MATERIALS AND METHODSCompound. MDL-860 was synthesized by L. Markley of The Dow Chemical Company, Midland, Mich. For easier dissolution in aqueous cell culture medium, the compound is first dissolved in 0.05 ml of dimethyl sulfoxide per 3 mg of MDL-860 and then diluted more than 100-fold in aqueous cell culture medium to produce a suspension of small particles.Viruses. Rhinovirus (RV) types 5, 8, 64, and Hank and RV types 1-A and 2, obtained from J. Gwaltney

“…Photosensitive HRV 1B was propagated in HeLa cells for more than three serial passages in the dark in the presence of 10 ,ug of neutral red per ml, as described by Mandel (12).…”

Section: Methodsmentioning

confidence: 99%

Mechanism of action of the antirhinovirus flavanoid 4',6-dicyanoflavan

Conti

Tomao

Genovese

et al. 1992

4',6-Dicyanoflavan (DCF), a new antirhinovirus compound, was shown to inhibit an early event of rhinovirus type 1B replication in HeLa cells. When DCF was present from the beginning of infection or was added no later than the first hour of infection, the compound completely prevented viral RNA and protein synthesis and the virus-induced shutoff of host translation. DCF had'no adverse effect either on virus binding to the cell membrane or on virus penetration into cells, whereas it delayed the uncoating kinetics of neutral redencapsidated rhinovirus. DCF also prevented mild acid or thermal inactivation of virus infectivity, although it reversibly interacted with virions. These results suggest that the stabilizing effect of DCF on virion capsid conformation is responsible for uncoating inhibition.Flavanoids are among the most potent inhibitors of human rhinovirus (HRV) replication. In the last few years, we have synthesized new anti-HRV flavanoids that are structurally related to the well-known flavanoid 4',6-dichloroflavan (BW683C) (1). An interesting feature of the synthesized flavanoid compounds is' that, besides having an effect on HRV (2), they exhibit an antiviral spectrum that is wider than that of BW683C. They show good antiviral activity against poliovirus type 2 (4), coxsa'ckievirus type B4, echovirus type 6, enterovirus type 71 (8), hepatitis A virus (18), and astrovirus (17). Studies on the mechanism of action indicate that they affect some early process of the virus life cycle (5), similar to the effect of BW683C (20).Recently, in an attempt to improve their antiviral potency, we synthesized new flavans, isoflavans, and isoflavenes substituted with halogens as well as cyano or amidino residues (3). The cyano residue is a strong electron-withdrawing group known to increase the antipicornavirus activity of substituted phenoxybenzene (13); the amidino group is present in some proteinase inhibitors with antiviral activity (6,19). Among these drugs, 4',6-dicyanoflavan (DCF) proved to be more active than the reference molecule BW683C when they were tested under the same experimental conditions (3). DCF had no adverse effect on cell cultures up to a concentration of 40 ,uM, which is about 1,740 times the 50% inhibitory concentration (IC50) of HRV type 1B plaque formation (IC50 = 0.023 ,uM).The aim of the present work was to elucidate whether the replacement of chlorine with cyano groups could influence the mechanism of the antiviral action of this type of molecule. MATERIALS AND METHODSCells and virus. The HeLa (Ohio) cell line was routinely grown in Eagle's minimum essential medium (MEM) as described previously (5).HRV type 1B (HRV 1B) was propagated in HeLa cells as reported previously (5). The virus titer was measured by plaque assay (2). * Corresponding author.Radioactively labeled virus was grown in the presence of 10 p,Ci of 5-[3H]uridine (27 Ci/mmol; Amersham International) per ml and actinomycin D (0.1 ,ug/ml). The preparation was digested with 1 mg of RNase A per ml (30 min, 33°C) to remove nonencap...