Tim Skern - Academia.edu (original) (raw)

Papers by Tim Skern

Protein Expression and Purification, 2014

The positive strand RNA genome of picornaviruses, including human rhinovirus (HRV), poliovirus (P... more The positive strand RNA genome of picornaviruses, including human rhinovirus (HRV), poliovirus (PV) and foot-and-mouth disease virus, is translated immediately into a polyprotein that is cleaved by virally encoded proteinases into 10-13 mature proteins. These include the four proteins required to assemble the viral particle as well as 3D(pol) (the viral RNA polymerase) and 2C, an ATPase and putative helicase. 2C is a protein which is responsible, together with 2B and 3A, for anchoring the replication complexes to membranous structures in the infected cell on which RNA replication takes place. Additionally, expression of 2C and its precursor 2BC in mammalian cells leads to vesicle formation observed in infected cells. 2C is encoded by all picornaviruses; nevertheless, its exact role in viral replication remains unclear. A contributing factor is the absence of structural data for this hydrophobic protein the generation of which has been hampered by an inability to produce soluble and stable material. Here, we compare 2C from several genera and show that the 2C protein has considerable heterogeneity. Using protein structure meta-analysis, we developed models of HRV14 2C that should be useful for mutational analysis. Based on these analyses, we expressed and purified two domains of HRV14 2C using three different protocols and examined the folding by thermal denaturation or (1)H NMR. Both domains were concentrated sufficiently to allow crystal screens or NMR pilot experiments to be performed. This work provides a platform to explore 2C proteins from all picornaviral genera to generate candidates for structural analysis.

The family Picornaviridae currently consists of 23 species in 9 genera (Enterovirus, Rhinovirus, ... more The family Picornaviridae currently consists of 23 species in 9 genera (Enterovirus, Rhinovirus, Cardiovirus, Aphthovirus, Hepatovirus, Parechovirus, Erbovirus, Kobuvirus and Teschovirus). Three new taxonomic proposals have been approved by the ICTV Executive Committee and await ratification by the ICTV membership. They are: i) to combine the enterovirus and rhinovirus genera, keeping the existing name Enterovirus; ii) to combine the species' Poliovirus and Human enterovirus C, retaining the latter name; and iii) to assign Human enterovirus C as the type species of the enterovirus genus. Two further proposals are in preparation: i) to create a new genus, Sapelovirus, consisting of three species, Porcine enterovirus A, Simian virus 2 and duck picornavirus TW90A (each to be renamed); and ii) to create a new unassigned species, Seneca Valley virus. Overall this will leave the number of genera unchanged, but result in the addition of two species. A number of issues are also being considered by the Study Group: i) the genus placement of Seneca Valley virus; ii) the position of avian encephalomyelitis virus as a tentative member of the genus Hepatovirus; iii) the proposal to place duck hepatitis virus type 1 (for which a number of genome sequences have recently been reported) in a new genus; iv) the proposal that bovine rhinovirus type 2 is classified as a new species in the genus Aphthovirus; and v) the proposal to divide bovine enteroviruses into two species. The Picornaviridae Study Group has a new website: www.picornastudygroup.com where the latest classification and proposals may be viewed.

Journal of General Virology, 2003

Mouse monoclonal antibodies (mAbs) were employed to select neutralization escape mutants of equin... more Mouse monoclonal antibodies (mAbs) were employed to select neutralization escape mutants of equine rhinitis A virus (ERAV). Amino acid changes in the ERAV mutants resulting in resistance to neutralization were identified in capsid protein VP1 at Lys-114, Pro-240 and Thr-241. Although the changes were located in different parts of the polypeptide chain, these mutants exhibited cross-resistance against all four mAbs employed, indicating that these residues contribute to a single immunogenic site. To explain this result, we constructed a model of the three-dimensional structure of the ERAV capsid based on comparison with the closely related foot-and-mouth disease virus (FMDV O 1 ). According to this model, VP1 is folded so that Lys-114 is in the bE-bF loop of the polypeptide chain at a considerable distance from Pro-240 and Trp-241 in the C-terminal region. However, around the fivefold axis of symmetry, the C terminus of VP1 in each protomer extends to the bE-bF loop of the adjacent VP1 in the next protomer. We therefore propose that the immunogenic site in ERAV is formed as a result of the close proximity of the Lys-114 residue in the bE-bF loop of one VP1 molecule and of the Pro-240/Thr-241 residues in the adjacent VP1 polypeptide chain. In terms of the overall architecture of the viral capsid structure, this site in ERAV most closely resembles the immunogenic site 1 of FMDV O 1 .

Virology, 1996

During the replication of rhino- and enteroviruses, the translation initiation factor elF-4 gamma... more During the replication of rhino- and enteroviruses, the translation initiation factor elF-4 gamma is specifically cleaved by the virally encoded 2 A proteinase. This cleavage has been proposed to lead to the inability of the host cell to translate its own capped mRNA and to stimulate internal initiation of protein synthesis from the viral mRNA. However, a direct causal relationship between these effects and 2A proteinase-mediated cleavage of elF-4 gamma has remained difficult to prove, mainly because of the toxicity of the 2A proteinase in mammalian expression systems. As an alternative approach, we placed the cDNA sequences for the human rhinovirus 2 2A proteinase and two mutants defective in proteolytic activity under the control of an inducible yeast Gal1-10 promoter and stably integrated them into the yeast genome. Induction of the wildtype enzyme led to changes in cellular morphology, an inhibition of cell division activity, and finally to cell death. As the yeast homologues of...

The Biochemical journal, Jan 15, 1996

The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to ser... more The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130-->Tyr almost completely inhibited enzyme activity at 37 degrees C; activity was, however, partially restored by the following exchanges: Ser-27-->Pro, His-135-->Arg or His-137-->Arg. To investigate this phenotypic reversion, 2A proteinases with the mutations Phe-130-->Tyr, Phe-130-->Tyr/His-135-->Arg, Phe-130-->Tyr/His-137-->Arg, His-135-->Arg or His-137-->Arg were expressed in Escherichia coli and purified. None of these mutations affected the affinity of the enzyme for a peptide substrate. However, the temperature-dependence of enzyme activity, as assayed by cleavage of a peptide substrate and by monitoring the toxicity of the proteinases towards the E. coli str...

Journal of virology, 1995

Rhinovirus and enterovirus 2A proteinases stimulate translation initiation driven from the cognat... more Rhinovirus and enterovirus 2A proteinases stimulate translation initiation driven from the cognate internal ribosome entry segment (IRES) (S. J. Hambidge and P. Sarnow, Proc. Natl. Acad. Sci. USA 89:10272-10276, 1992; H.-D. Liebig, E. Ziegler, R. Yan, K. Hartmuth, H. Klump, H. Kowalski, D. Blaas, W. Sommergruber, L. Frasel, B. Lamphear, R. Rhoads, E. Kuechler, and T. Skern, Biochemistry 32:7581-7588, 1993). Given the functional similarities between the foot-and-mouth disease virus (FMDV) L proteinase and these 2A proteinases (autocatalytic excision from the nascent viral polyprotein and cleavage of eIF-4 gamma), we investigated whether the FMDV L proteinase would also be able to stimulate translation initiation. We found that purified recombinant FMDV Lb proteinase could stimulate in vitro translation driven from a rhinovirus or enterovirus IRES by 5- to 10-fold. In contrast, stimulation of translation initiation on a cardiovirus IRES by this proteinase was minimal, and stimulation ...

$Research paper thumbnail of Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus$

Virus Research, 2014

Cross-reactions observed in serological assays between Usutu virus (USUV), the USUV outlier subty... more Cross-reactions observed in serological assays between Usutu virus (USUV), the USUV outlier subtype strain CAR_1969 and West Nile virus (WNV) suggest that they share antigenic features amongst their structural outer proteins especially envelope (E) proteins. To investigate the molecular background of this observation, we compared the E protein sequences of seven USUV strains, USUV subtype strain CAR_1969 and WNV strain 2471, focusing on the binding site defined by the WNV neutralizing antibody E16. USUV SouthAfrica_1959 differs from WNV 2741 in three of four residues critical for E16 antibody binding and five of the 12 additionally involved residues. In contrast, USUV subtype CAR_1969 differs from WNV 2741 in two critical residues and five additional residues. Furthermore, USUV subtype CAR_1969 differs from other USUV strains in two critical residues. E16 antibody binding has previously been shown to be highly specific for WNV; thus, the observed variation in amino acid residues suggests that the region corresponding to the WNV E16 epitope is probably not responsible for the observed cross-reactions between WNV and USUV. Seroneutralisation assays confirmed these findings for WNV and USUV, however, showed occurring cross-reactivity between WNV and USUV subtype CAR_1969 at high antibody titers. The sequence diversity in this region might also explain some of the observed different antigenic characteristics of USUV strains and USUV subtype CAR_1969. A therapeutic effect of E16 antibody has been described in WNV infected mice; therefore, a USUV specific antibody generated against the region corresponding to the WNV E16 binding site might represent an approach for treating USUV infections.

Virus Research, 1999

Picornaviruses are a family of positive-strand RNA viruses the members of which include polioviru... more 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. : S 0 1 6 8 -1 7 0 2 ( 9 9 ) 0 0 0 4 3 -X

Molecular Biology of Picornavirus, 2002

FEBS Letters, 2014

The leader protease (L(pro)) from foot-and-mouth disease virus (FMDV) has the ability to cleave e... more The leader protease (L(pro)) from foot-and-mouth disease virus (FMDV) has the ability to cleave eIF4G, leading to a blockade of cellular protein synthesis. In contrast to previous reports, our present findings demonstrate that FMDV L(pro) is able to increase translation driven by FMDV IRES. Additionally, inactivation of eIF2 subsequent to phosphorylation induced by arsenite or thapsigargin in BHK cells blocks protein synthesis directed by FMDV IRES, whereas in the presence of L(pro), significant translation is found under these conditions. This phenomenon was also observed in cell-free systems after induction of eIF2 phosphorylation by addition of poly(I:C).

Virus Research, 2009

Equine rhinitis A virus (ERAV) is a picornavirus which causes an acute respiratory infection in h... more Equine rhinitis A virus (ERAV) is a picornavirus which causes an acute respiratory infection in horses worldwide, and virus neutralization (VN) has been the standard method for the detection of ERAV antibody in horse serum. Previous studies have identified recombinant virion protein VP1 (rVP1) purified under native conditions to be of high potential for the development of a diagnostic ERAV enzyme-linked immunosorbent assay (ELISA). This study presents an optimized protocol for the expression and purification of native full-length rVP1. Furthermore, we demonstrate that, upon denaturation, rVP1 no longer reacts to ERAV antibody in a prototype ELISA. Size exclusion chromatography (SEC) performed on native and denatured rVP1 indicates that denatured rVP1 forms multimeric aggregates that may causally connect to the loss of antigenicity observed in the ELISA.

Virology, 1997

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Z... more 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. ᭧ 1997 Academic Press

Veterinary Microbiology, 2005

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infec... more Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

European Journal of Biochemistry, 1996

Tissue-polypeptide-specific antigen (TPS) from the human colon adenocarcinoma cell line WiDr was ... more Tissue-polypeptide-specific antigen (TPS) from the human colon adenocarcinoma cell line WiDr was purified using the monoclonal antibody M3 as a probe. Upon SDS/polyacrylamide gradient gel electrophoresis, several TPS-positive bands were detected (corresponding to 13 kDa, 22 kDa and a doublet at 42 kDa). The 13-kDa moiety was purified about 30 000-fold by a 5-step protocol. The electro-phoretically homogeneous component was obtained in a 7% yield of the total TPS activity of the crude extract. Nterminal sequence analysis showed the presence of an N-terminally truncated molecule and identified the 13-kDa TPS component as a fragment of human cytokeratin 18, with a major form starting at position 284 of the parent molecule. Laser-desorption mass spectrometry showed the presence of one major component with a molecular mass corresponding to a C-terminal end close to position 396 (which gives 12776Da for the form with non-truncated N-terminus). The M3 antibody was also used to screen a human prostate cDNA I g t l l library. Four identical phage clones were detected, each producing a fusion protein with P-galactosidase and the M3-positive component. PCR amplification showed the presence of an approximately 1200-bp insert, and sequence analysis revealed it to contain a 996-nucleotide fragment corresponding to residues 103-429 of human cytokeratin 18 (plus a non-coding human desmin artifact fragment). Smaller fragments, engineered by PCR and expressed as fusion proteins using the pET3xc vector in Escherichia coli, showed that the M3 epitope is localized to cytokeratin 18, residues 322-340. Two other TPS-active monoclonal antibodies were localized to cytokeratin 18 with similar techniques, ascribing an epitope (to M21) to residues 414-429 and another (to M24) to residues 139-297. Combined, the results demonstrate that TPS reactivity is derived from specific epitopes of human cytokeratin 18.

Proceedings of the National Academy of Sciences, 1974

The structural analysis of the Fab' fragment of human myeloma immunoglobulin IgGl(lambda)... more The structural analysis of the Fab' fragment of human myeloma immunoglobulin IgGl(lambda) New has been extended to a nominal resolution of 2.0 A. Each of the structural subunits corresponding to the variable and to the constant homology regions of the light and heavy chains contains two irregular beta-sheets which are roughly parallel to each other and surround a tightly packed interior of hydrophobic side chains. About 50-60% of the amino-acid residues are included in beta-pleated sheets. Sequence alignments between the homology regions of Fab' New obtained by comparison of their three-dimensional structures are given. Some of the sequence variations observed in light and heavy chains and the role of the regions of hypervariable sequence in defining the size and shape of the active site of different immunoglobulin molecules are discussed on the basis of the three-dimensional model of Fab' New.

Proceedings of the National Academy of Sciences, 1987

The complete nucleotide sequence of the genome of human rhinovirus type 89 was determined from th... more The complete nucleotide sequence of the genome of human rhinovirus type 89 was determined from the cDNA that had been cloned into Escherichia coli. The genome is 7152 nucleotides long and contains a single large open reading frame of 2164 codons. Translation commences at position 619 and ends 42 nucleotides before the poly(A) tract. The positions of three proteolytic cleavage sites in the polyprotein were determined by N-terminal amino acid sequencing of the capsid proteins; the remainder were predicted from comparisons with other picornaviruses. Extensive similarity between the derived amino acid sequences of human rhinovirus types 89 and 2 was found, whereas the similarity between human rhinovirus types 89 and 14 was considerably less. It is apparent that human rhinoviruses may be more closely related than has been previously thought.

Nucleic Acids Research, 1985

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to d... more cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5'end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3' half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5' half (including the capsid proteins) are more heterogeneous.