Biomedical subjects

J Seipelt

Publications and source records attributed to J Seipelt.

7 recordsLinked to original sources

PubMed2005

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

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.

Antioxidants↗

PubMed2004

Molecular cloning, expression and purification of protein 2A of hepatitis A virus.

Expression of the protein 2A of Hepatitis A virus (HAV), spanning amino acids 764 through 981 of the viral polyprotein results in a strong inhibition of cap-dependent translation (Maltese et al., 2000). However, the molecular mechanism responsible has remained unclear, in part because the HAV 2A protein was not available in amounts large enough to allow biological or structural studies. To address this issue, a cDNA representation of the sequences encoding HAV 2A was generated by PCR, using primers that introduced an AUG triplet, and a sequence coding for 6 histidine residues at the 5'- and 3'-termini of the genomic sequence, respectively. The cDNA fragment was introduced by cassette exchange in the inducible expression vector pQE-60, and the construct was propagated in bacteria E. coli M15 which constitutively expresses the lac repressor. Upon induction with IPTG (1 mM), HAV 2A was visualized by SDS-PAGE of bacterial lysates as a prominent band M(r) = 21 kDa. The identity of the polypeptide was confirmed by both MALDI-TOF peptide mapping and direct amino acid sequencing. The His-tagged HAV 2A was extracted from bacterial pellets under totally denaturing conditions (6 M urea), subjected to Ni(++)-Sepharose affinity chromatography, allowed to refold while still attached to the matrix, and eluted with 250 mM Imidazole. Contaminant material was partly removed by differential ammonium sulfate precipitation. The protein was further concentrated (Vivaspin centrifugal concentrator), the insoluble material (if present) was discarded, and the homogeneity of the dispersion was ascertained by light scattering. SDS-PAGE revealed that in addition to the main protein (Mr = 21 kDa), a second one of apparent Mr = 14 kDa was always present in variable amounts. The proportion of the latter tended to increase with aging of the preparation. Edman degradation analysis proved that the 14 kDa protein resulted from the cleavage of HAV 2A at a so far undetected scissile bond Gly856/Val857 of the viral polyprotein. A first attempt to crystallize the protein by the hanging drop procedure yielded only small crystals containing exclusively the 14 kDa derivative of HAV 2A. Western blot analysis of HeLa cell extracts that had been incubated with the His-tagged HAV 2A so purified failed to reveal any change in the electrophoretic mobility of the eukaryotic initiation factor (eIF) 4G I.

Amino Acid Sequence↗

PubMed2000

2A proteinase of human rhinovirus cleaves cytokeratin 8 in infected HeLa cells.

Rhino- and enteroviruses encode two proteinases, 2A and 3C, which are responsible for the processing of the viral polyprotein and for cleavage of several cellular proteins. To identify further targets of the 2A proteinase of human rhinovirus serotype 2 (HRV2), an in vitro cleavage assay followed by two-dimensional electrophoresis was employed. Cytokeratin 8, a member of the intermediate filament group of proteins, was found to be proteolytically cleaved in vitro by the 2A proteinase of HRV2 and of coxsackievirus B4 and in vivo during HRV2 infection of HeLa cells. The cleavage results in removal of 14 amino acids from the N-terminal head domain of cytokeratin 8. However, other intermediate filament proteins (cytokeratins 7 and 18 and vimentin) were not cleaved in the course of the HRV2 infection. Compared with the processing of the eucaryotic translation initiation factors 4GI and 4GII, cleavage of cytokeratin 8 occurs late in the infection cycle at the time of the onset of the cytopathic effect.

Blotting, Western↗

PubMed1999

The structures of picornaviral proteinases.

Picornaviruses are a family of positive-strand RNA viruses the members of which include poliovirus, hepatitis A virus, rhinovirus, foot-and-mouth disease virus and encephalomyocarditis virus. The genetic information contained in the single-stranded, positive sense RNA genome is expressed as a single protein of around 2000 amino acids. This primary product of protein synthesis, designated the polyprotein, is subsequently cleaved into the mature viral proteins by proteinases present within it. The properties of the three defined proteolytic activities present in the picornaviruses are reviewed and the three-dimensional structures of the hepatitis A 3C proteinase and the leader proteinase of foot-and-mouth disease virus as well as a model of the structure of the HRV2 2A proteinase are compared with those of chymotrypsin, papain and streptomyces griseus A proteinase, respectively.

3C Viral Proteases↗

PubMed1997

Mutational analyses support a model for the HRV2 2A proteinase.

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity. A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase. The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular ("cis") and intermolecular ("trans") activities. The HRV2 proteinase 2A is proposed to have a two domain structure, with the catalytic site and substrate binding region on one face of the molecule and a Zn-binding motif on the opposite face. Residues Gly 123, Gly 124, Thr 121, and Cys 101 are proposed to be involved in the architecture of the substrate binding pocket and to provide the correct environment for the catalytic triad of His 18, Asp 35, and Cys 106. Residues Tyr 85 and Tyr 86 are thought to participate in substrate recognition. The presence of an extensive C-terminal helix, in which Asp 132, Arg 134, Phe 130, and Phe 136 play important roles, explains why mutations in this region are generally detrimental to proteinase activity. The proposed Zn-binding motif comprises Cys 52, Cys 54, Cys 112, and His 114. Exchange of these residues inactivates the enzyme. Furthermore, as measured by atom emission spectroscopy, Zn was absent from purified preparations of proteinase 2A in which His 114 had been replaced by Asn. The absence of disulphide bridges was confirmed by subjecting highly purified HRV2 proteinase 2A to one- and two-step alkylation procedures.

Amino Acid Sequence↗

PubMed1994

The 2A proteinase of human rhinovirus is a zinc containing enzyme.

The 2A proteinase of human rhinovirus (HRV) 2 is a cysteine proteinase which is not inhibited by metal chelating agents. However, total hydrolysis of highly purified HRV2 2A followed by atom emission spectroscopy demonstrated that HRV2 2A contains 1 mol of zinc per mole of enzyme. Furthermore, Zn depletion of the enzyme led to a loss of proteolytic activity which could subsequently be restored by Zn supplementation.

Amino Acid Sequence↗

PubMed1994

2A proteinases of coxsackie- and rhinovirus cleave peptides derived from eIF-4 gamma via a common recognition motif.

The cleavage specificities of the 2A proteinases from coxsackievirus B4 (CVB4) and human rhinovirus 2 (HRV2) on oligopeptide substrates have been determined. Comparison of the specificity of CVB4 2A proteinase with that of HRV2 2A proteinase allowed cleavable peptides to be designed using the common motif IIe/Leu-X-Thr-X*Gly; little resemblance to the viral cleavage site remained. The data also allowed the prediction of three possible cleavage sites for 2A proteinases on eIF-4 gamma; two peptides derived from these sequences were cleaved by both 2A proteinases. One of these peptides corresponds to the cleavage site for 2A proteinases mapped on eIF-4 gamma [B. J. Lamphear et al. (1993) J. Biol. Chem. 268, 19200-19203]. This supports the hypothesis that cleavage of eIF-4 gamma by picornaviral 2A proteinases occurs directly.

Amino Acid Sequence↗