Inhibition of polyprotein processing and RNA replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions

Abstract

Pyrrolidine dithiocarbamate (PDTC) is an antiviral compound that was shown to inhibit the replication of human rhinoviruses (HRVs), poliovirus, and influenza virus. To elucidate the mechanism of PDTC, the effects on the individual steps of the infection cycle of HRV were investigated. PDTC did not interfere with receptor binding or internalization by receptor mediated endocytosis of HRV2 particles into HeLa cells. But we demonstrate that the processing of the viral polyprotein was prevented by PDTC treatment in HeLa cells infected with HRV2. Furthermore, PDTC inhibited the replication of the viral RNA, even when added four hours post infection. As PDTC is described as a metal ion binding agent, we investigated the effect of other metal chelators on the multiplication of HRV2. We show that EDTA, omicron-phenanthroline, and bathocuproine disulfonic acid do not exhibit any antiviral properties. Surprisingly, these substances, coadministered with PDTC, abolished the antiviral effect of PDTC, suggesting that metal ions play a pivotal role in the inhibition of virus multiplication. These results suggest that PDTC inhibits the activity of the viral proteases in a metal ion dependent way.

FIG. 1.

PDTC does not inactivate or interact with the virus capsid. HRV2 virus was preincubated with 125 μM PDTC for 1 h. HeLa cells were infected with pretreated or untreated HRV2 (multiplicity of infection, 20) for 1 h at 4°C. Unbound virus was washed away and cells were incubated in infection medium supplemented with 125 μM PDTC where indicated at 37°C. Virus titers of the supernatants were determined by TCID₅₀ assay after 24 h.

FIG. 2.

PDTC does not interfere with internalization of HRV2. HeLa cells grown on coverslips were infected with HRV2 (multiplicity of infection, 50) in the absence or presence of 125 μM PDTC. Cells were fixed using 4% paraformaldehyde 20 min, 60 min, and 8 h postinfection The virus capsid protein VP2 was localized by the monoclonal antibody 8F5 and visualized by immunofluorescence.

FIG. 3.

PDTC has no major effect on IRES dependent translation. At 6 h p.i. the level of IRES-dependent protein synthesis in HeLa cells, infected with HRV2 (multiplicity of infection, 100), was determined by metabolic labeling for 1 h; 125 μM PDTC or 125 μM cycloheximide (CHX) was added 15 min before the labeling and present during the period of the pulse. The amount of incorporated [³⁵S]methionine/cysteine was determined by protein precipitation and measurement by scintillation counting. Error bars indicate the standard deviation from experiments carried out in triplicate.

FIG. 4.

PDTC interferes with the polyprotein processing. (A) Viral proteins in HeLa cells, infected with HRV2 (multiplicity of infection 100), were labeled by incorporation of [³⁵S]methionine/cysteine for 1 h starting 6 h 30 min p.i. in the presence of 125 μM PDTC or 200 μM zVAD.fmk. Cell lysates were prepared and analyzed by SDS-PAGE and autoradiography. (B) HeLa cells, infected with HRV2 (multiplicity of infection, 50), were preincubated with PDTC for 30 min and subsequently incubated in medium supplemented with [³⁵S]methionine/cysteine and 125 μM PDTC to label viral proteins for 60 min starting 6 h p.i. Then the cells were transferred to infection medium containing 125 μM PDTC. Cell lysates were prepared every hour. VP2 or VP2 comprising precursor proteins were isolated by immunoprecipitation employing the specific antibody 8F5 and analyzed by SDS-PAGE and autoradiography. Radiolabeled HRV2 was separated in parallel to identify viral capsid proteins (*HRV2). Mock designates immunoprecipitation from uninfected but labeled cells. (C) Viral proteins were labeled in virus-infected HeLa cells, infected with HRV2 (multiplicity of infection 50), for 15 min starting 6 h 45 min p.i. Subsequently the cells were treated with 125 μM PDTC or 125 μM cycloheximide for 2 h. Cell lysates were prepared and proteins were analyzed by SDS-PAGE and autoradiography.

FIG. 5.

PDTC decreases the replication of positive and negative strand RNA of HRV2. HeLa cells were infected with HRV2 (multiplicity of infection, 20). At the start of viral RNA replication at 4 h p.i. PDTC was added where indicated (+). Total RNA was isolated 1, 2, 4, 6, 8, and 13 h p.i. The level of positive- and negative-strand HRV2 RNA was quantitated by Northern blot analysis using strand-specific probes generated by primer extension in the presence of [γ-³²P]dCTP and visualized by autoradiography. Analysis of the 28S rRNA ensured an equal loading.

FIG. 6.

Antiviral effect of PDTC is abolished by metal chelators. HeLa cells, infected with HRV2 (multiplicity of infection 20), were treated with the metal chelators BCS (A), EDTA (B), or ο-phenanthroline (C). 125 μM PDTC were added simultaneously. The virus titers in the supernatant of the infected cells were determined by TCID₅₀ assay 24 h p.i.

FIG. 7.

Zinc and copper ions are involved in the antiviral effect of PDTC. HeLa cells, infected with HRV2 (multiplicity of infection, 20), were treated with 125 μM PDTC and 2.5 μM EDTA (dark bars). Simultaneously, Zn²⁺ (A), Fe²⁺ (B), or Cu²⁺ (C) ions were added. The virus titers in the supernatant of the infected cells were determined by TCID₅₀ assay at 24 h p.i. Light bars represent cells treated with the respective ions but without PDTC and without EDTA.