The Processing of eIF4GI by Human Rhinovirus Type 2 2A pro : Relationship to Self-Cleavage and Role of Zinc

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, -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.Human rhinoviruses (HRVs) are the most frequent cause of the common cold and are implicated in more than 50% of upper respiratory tract infections (45). Although not life threatening, infection with HRV can prepare the ground for more serious diseases, such as acute exacerbation of asthma (20, 30) or otitis media (5). As there are more than 100 HRV serotypes, vaccine development is unfeasible. Apart from symptomatic medication no causative treatment for HRV infections is currently available. Therefore, analysis of functions of new antiviral substances is of great interest and might also shed light on novel aspects of virus-cell interactions.HRVs belong to the genus Picornaviridae. These viruses have a single-stranded positive-sense RNA genome of approximately 7,400 nucleotides. The viral RNA encodes four capsid proteins (VP1 through VP4) and seven nonstructural proteins that are involved in viral RNA replication and polyprotein processing. The HRV serotypes can be classified into A and B groups based on sequence alignments (36). Alternatively, HRVs are divided into two groups according to their receptor specificity (1, 49). Members of the major group HRVs bind to the intercellular adhesion molecule 1 (ICAM-1) (15, 44), whereas serotypes of the minor group use various members of the low-density lipoprotein receptor family (18).Upon receptor binding, the viral particle is internalized by receptor-mediated endocytosis. After acidification of the late endosome and subsequent uncoating, the RNA of minor group HRVs is released into the cytoplasm (32). Then the viral RNA is translated into a single large polyprotein from an internal ribosome entry site (IRES), which is located in the 5Ј untranslated region of the HRV genome. The polyprotein is processed into the mature viral proteins through a sequence of cleavages performed by two virus-encoded prot...

Section: Discussionmentioning

confidence: 85%

Inhibition of Polyprotein Processing and RNA Replication of Human Rhinovirus by Pyrrolidine Dithiocarbamate Involves Metal Ions

Krenn

Holzer

Gaudernak

et al. 2005

“…8C). To determine whether impairing eIF4F through another mechanism also stimulated IRES activity, we expressed the rhinovirus 2A protease (2A pro ), which cleaves eIF4G (19,22 (Fig. 8D).…”

Section: Analysis Of Crpv Ires Translation In Vitromentioning

confidence: 99%

Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

Garrey

Lee

et al. 2010

104

The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2␣ phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2␣ by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2␣ phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2␣ phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5 untranslated region (5UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5UTR and IGR IRES.

“…An important cellular substrate for 2A pro is eIF4G (5,37,38). Cleavage of this translation protein leads to inhibition of host protein synthesis during picornavirus infection (1,5,22,63,68). To determine whether translation inhibition plays a role in the ability of PV to replicate in cells treated with IFN-␣, cellular protein synthesis was examined after labeling with [ 35 S]methionine.…”

Section: Replication Of Vsv Emcv and P1m In Ifn-␣-treated Hela Cellsmentioning

confidence: 99%

Proteinase 2A^proIs Essential for Enterovirus Replication in Type I Interferon-Treated Cells

Morrison

Racaniello

2009

The Picornaviridae family comprises a diverse group of small RNA viruses that cause a variety of human and animal diseases. Some of these viruses are known to induce cleavage of components of the innate immune system and to inhibit steps in the interferon pathway that lead to the production of type I interferon. There has been no study of the effect of picornaviral infection on the events that occur after interferons have been produced. To determine whether members of the Enterovirus genus can antagonize the antiviral activity of interferon-stimulated genes (ISGs), we pretreated cells with alpha interferon (IFN-␣) and then infected the cells with poliovirus type 1, 2, or 3; enterovirus type 70; or human rhinovirus type 16. We found that these viruses were able to replicate in IFN- ␣