Elsevier

Antiviral Research

Volume 67, Issue 2, August 2005, Pages 98-106
Antiviral Research

Ion transport blockers inhibit human rhinovirus 2 release

https://doi.org/10.1016/j.antiviral.2005.05.003Get rights and content

Summary

Picornavirus replication causes leakage of cytoplasmic K+ and an influx of Na+ and Ca2+. In this study, we have explored the possibility that a blockade of Ca2+ and Na+ influx would reduce rhinovirus production and/or release. The Ca2+-channel blockers, verapamil and diltiazem, as well as the blocker of Na+/H+ exchange and the epithelial Na+ channel, EIPA, inhibited both virus production and release. The effect on virus release was more pronounced than the effect on production, thus raising the possibility that rhinovirus release may serve as a target for antiviral agents. Unexpectedly, our results also showed that the antiviral activity of the Ca2+-channel blockers was not due to the block of Ca2+ influx. Similarly, the antiviral activity of EIPA appeared to be unrelated to the blockade of cellular Na+/H+ exchanger or the epithelial Na+ channel. Potential alternative mechanisms of the antiviral activity of these compounds are discussed.

Introduction

The Picornaviridae are a family of non-enveloped animal viruses. They are currently divided into 9 genera, three of which are significant causes of human disease, the Enteroviruses, Rhinoviruses and Hepatoviruses. To date, no antiviral agent has been approved for the treatment of these infections. Drug development has been targeting steps of the replication cycle such as virus entry, RNA replication, protein synthesis and cleavage, and virion assembly. Here, we present evidence that progeny virus release from infected cells may have the potential to serve as an antiviral target.

It is assumed that the release of picornavirus progeny occurs via cell lysis, with the exception of hepatitis A virus. The mechanism of this process is largely unknown. Studies conducted on the Enteroviruses and Cardioviruses have demonstrated alterations in transport of ions and small molecules across the plasma membrane of infected cells, coinciding with virus production. Virus replication causes leakage of cytoplasmic K+ and influx of Na+ into the cytoplasm (Carrasco and Smith, 1976, Egberts et al., 1977, Lopez-Rivas et al., 1987, Nair, 1981, Nair et al., 1979, Schaefer et al., 1982). At the same time Ca2+ content of the ER and the Golgi is reduced and an influx of extracellular Ca2+ into the cytoplasm takes place (Irurzun et al., 1995; van Kuppeveld et al., 1997a). These events result in the collapse of ion gradients across the plasma membrane, and Na+ and Ca2+ overload of infected cells. In addition to ions, non-permeant molecules under 750 Da become permeant whereas molecules over 10 kDa remain impermeant (Carrasco, 1978, Contreras and Carrasco, 1979). A hypothesis has been formulated that the changes in membrane permeability lead to cell lysis and release of virus progeny (Carrasco, 1995), which is partly supported by the data showing that the increase in intracellular Ca2+ concentration ([Ca2+]i) facilitates enterovirus release (van Kuppeveld et al., 1997a).

We have explored the possibility that blockade of Ca2+ and Na+ influx would reduce rhinovirus production and/or release. The blockers of Ca2+ channels, verapamil and diltiazem, and the blocker of Na+/H+ exchange and the epithelial Na+ channel, EIPA, inhibited both rhinovirus 2 production and release. However, the antiviral activity of these compounds was not due to the blockade of Ca2+ influx, or Na+ influx via Na+/H+ exchanger or the epithelial Na+ channel.

Section snippets

Chemicals

Ion transport inhibitors were purchased from Sigma. Verapamil, (+)-cis-diltiazem and benzamil were dissolved in water; 5-(N-ethyl-N-isopropyl)amiloride and nifedipine in ethanol; and amiloride in DMSO.

Cells and viruses

HeLa T cells (picornavirus-susceptible HeLa line) were obtained from Victorian Infectious Diseases Reference Laboratory (Melbourne, Australia) and maintained in minimal essential medium (MEM, Gibco), supplemented with 5% heat-inactivated fetal bovine serum (FBS, Gibco).

Human rhinoviruses 2 and 14

Ca2+ increase in HRV2-infected HeLa cells precedes membrane permeation to ethidium bromide

The increase in cell membrane permeability to cations and small molecules coinciding with virus production has been shown in studies conducted on enteroviruses and a cardiovirus. There are no published data regarding the membrane permeability of rhinovirus-infected cells. Therefore, we examined the effects of human rhinovirus 2 (HRV2) infection on the intracellular Ca2+ content of HeLa cells.

HeLa cells were infected with HRV2 at an MOI of 15 plaque-forming units (PFU) per cell, and

Discussion

Picornavirus replication causes leakage of cytoplasmic K+ and influx of Na+ and Ca2+ into the cytoplasm. In this study, we have explored the possibility that blockade of Ca2+ and Na+ influx would reduce rhinovirus production and/or release.

We have demonstrated that [Ca2+]i increase in HRV2-infected HeLa cells precedes plasma membrane permeation to ethidium bromide. This suggested that virus-induced cation fluxes may occur via cellular ion transport pathways, which could be blocked by ion

Acknowledgements

This work was supported in part by Select Vaccines Ltd., the Joe White Bequest, and NHMRC fellowship 149502 (D.A.). The authors are grateful to Damian Myers for helpful discussions.

References (27)

  • S. Diochot et al.

    Dihydropyridines, phenylalkylamines and benzothiazepines block N-, P/Q- and R-type calcium currents

    Pflugers Arch.

    (1995)
  • J.R. Doedens et al.

    Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A

    EMBO J.

    (1995)
  • E. Egberts et al.

    Alteration of the intracellular energetic and ionic conditions by mengovirus infection of Ehrlich ascites tumor cells and its influence on protein synthesis in the midphase of infection

    J. Virol.

    (1977)
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