Ernst Kuechler

Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses.

Equine rhinoviruses (ERVs) are picornaviruses which cause a mild respiratory infection in horses. The illness resembles the common cold brought about by rhinoviruses in humans; however, the presence of a viraemia during ERV-1 infection, the occurrence of persistent infections and the physical properties are all more reminiscent of foot-and-mouth disease virus (FMDV). cDNA cloning and sequencing of the genomes of ERV-1 and ERV-2 between the poly(C) and poly(A) tracts showed that the serotypes are heterogeneous. Nevertheless, the genomic architecture of both serotypes is most similar to that of FMDV. Indeed, a comparison of the derived protein sequences of ERV-1 shows that their identity is greatest to FMDV. In contrast, most ERV-2 proteins are more related to encephalomyocarditis virus (EMCV) proteins than they are to FMDV or ERV-1. These results place ERV-1 alongside FMDV in the aphthovirus genus of the picornavirus family and indicate that this virus may serve as a model system for examining the biology of FMDV.

The structure of the 2A proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis

The crystal structure of the 2A proteinase from human rhinovirus serotype 2 (HRV2‐2Apro) has been solved to 1.95 Å resolution. The structure has an unusual, although chymotrypsin‐related, fold comprising a unique four‐stranded β sheet as the N‐terminal domain and a six‐stranded β barrel as the C‐terminal domain. A tightly bound zinc ion, essential for the stability of HRV2‐2Apro, is tetrahedrally coordinated by three cysteine sulfurs and one histidine nitrogen. The active site consists of a catalytic triad formed by His18, Asp35 and Cys106. Asp35 is additionally involved in an extensive hydrogen‐bonding network. Modelling studies reveal a substrate‐induced fit that explains the specificity of the subsites S4, S2, S1 and S1′. The structure of HRV2‐2Apro suggests the mechanism of the cis cleavage and its release from the polyprotein.

Mechanism of entry of human rhinovirus 2 into HeLa cells.

Internalized human rhinovirus 2 (HRV2) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20 degrees but not at 34 degrees the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20 degrees prior to addition of monensin permits virus synthesis to occur, depending on the time of preincubation.

Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA.

Photo‐reactive 3‐(4′‐benzoylphenyl)propionyl‐Phe‐tRNA bound to the A‐ or the P‐site was crosslinked to 23S RNA upon irradiation at 320 nm. The sites of reaction were identified as U‐2584 and U‐2585 at the A‐site and A‐2451 and C‐2452 at the P‐site. Minor crosslinks from both sites were observed at nucleotides A‐2503 to U‐2506. All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V. Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo‐crosslinking. In addition, tetracycline was also shown to photo‐crosslink to this region. These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A‐ and P‐site.

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

Antiviral Effects of Pyrrolidine Dithiocarbamate on Human Rhinoviruses

ABSTRACT Human rhinoviruses (HRVs) are the predominant cause of the common cold. The frequency of HRV infections in industrial countries and the lack of effective therapeutical treatment underline the importance of research for new antiviral substances. As viral infections are often accompanied by the generation of oxidative stress inside the infected cells, several redox-active substances were tested as potential antivirals. In the course of these studies it was discovered that pyrrolidine dithiocarbamate (PDTC) is an extremely potent compound against HRV and poliovirus infection in cell culture. Besides the ability to dramatically reduce HRV production by interfering with viral protein expression, PDTC promotes cell survival and abolishes cytopathic effects in infected cells. PDTC also protects cells against poliovirus infection. These effects were highly specific, as several other antioxidants (vitamin C, Trolox, 2-mercaptoethanol, and N-acetyl-l-cysteine) are inactive against HRV infection. Synthesis of HRV proteins and cleavage of eucaryotic initiation factor 4G responsible for host cell shutoff of cellular protein synthesis are severely inhibited in the presence of PDTC.

Cap-recognizing protein of influenza virus

Abstract Globin mRNA-primed in vitro transcription of influenza virus is inhibited by m 7 GpppGm, m 7 GpppG, and m 7 GTP indicating that the cap analogs block the specific binding site involved in the cap transfer reaction. γ-[4-(Benzoylphenyl)methylamido]-7-methylguanosine-5′-triphosphate, a photoreactive derivative of m 7 GTP, was used as an affinity label for the cap-binding protein. Evidence for the specificity of the labeling reaction is provided by competition with various cap analogs. At low concentrations inhibition of affinity labeling was observed only with guanosine derivatives which also exert a strong inhibitory effect on in vitro transcription. The cap-binding protein was tentatively identified as P2 by electrophoresis on polyacrylamide gels.

A neutralizing epitope on human rhinovirus type 2 includes amino acid residues between 153 and 164 of virus capsid protein VP2.

Use has been made of a monoclonal antibody (designated 8F5) to map a neutralizing epitope on the viral capsid protein VP2 of human rhinovirus 2 (HRV2). This antibody which was raised against the native virus, neutralizes HRV2 and is also capable of recognizing denatured VP2 on Western blots. To examine the binding site of 8F5, VP2 of HRV2 was expressed in Escherichia coli. Deletions starting at the 3 end were then introduced into the gene for VP2 using Bal-31 nuclease. Polypeptides shortened at the carboxy terminus of VP2 were obtained from the deletions and were blotted onto nitrocellulose. The samples were then probed with monoclonal antibody 8F5. Recognition by 8F5 was maintained as long as the expressed polypeptide contained the VP2 sequence up to amino acid 164 or beyond. However, when the VP2 sequence was truncated to amino acid 153 or less 8F5 was no longer able to bind. The neutralization epitope (or part of it) recognized by 8F5 on VP2 is therefore located between amino acids 153 and 164.

Proteins at the mRNA binding site of the Eschericria coli ribosome

Factors involved in the binding of messenger RNA to ribosomes during initiation of protein synthesis have been studied extensively (for a review see [l] ), but less is known about the role of the ribosome itself in these processes. Several lines of evidence indicate that protein Sl of the 30s ribosomal subunit is involved in the binding of poly U as well as natural messenger RNA [2-91 Recently we have described experiments which prove that protein Sl is in close contact with the messenger RNA on the ribosome [lo] . For these experiments the polynucleotide poly(s4 U)‘, which can be photoactivated, was employed as messenger RNA. This polynucleotide was obtained by enzymatic polymerization of 4-thiouridine-diphosphate with I?. coli polynucleotide phosphorylase [ 111. The polymer not only resembles poly U in its physicochemical properties [ 111 but it can also serve. as messenger RNA in an in vitro protein synthesizing system. At 20 mM Mg2+ concentration poly(s4U) stimulates binding of Phe-tRNA to E. coli ribosomes and codes for the synthesis of poly Phe ([ 121, and in preparation). The 4-thiouracil ring can be photoactivated at 330 nm to react with proteins [ 131. The activity of the ribosome itself is not affected by irradiation at this wavelength [lo] . [3 H] poly(s4U) was bound to

STABILIZATION OF HUMAN RHINOVIRUS SEROTYPE 2 AGAINST PH-INDUCED CONFORMATIONAL CHANGE BY ANTIVIRAL COMPOUNDS

Four WIN compounds with anti-picornavirus activities were tested for their ability to stabilize human rhinovirus serotype 2 (HRV-2) against low pH-induced conformational changes in vitro, as determined by specific immunoprecipitation. These results were compared to the minimal inhibitory concentration (MIC) as measured in a plaque reduction assay. A direct relationship was observed between the concentration of the compound that prevented the low pH-induced conformational changes and the MIC, indicating that stabilization is an important element in the mode of action of these drugs against HRV-2.