A single amino acid substitution in viral VP1 protein alters the lytic potential of clone-derived variants of echovirus 9 DM strain in human pancreatic islets.

In vitro studies with primary human pancreatic islets suggest that several enterovirus serotypes are able to infect and replicate in beta cells. Some enterovirus strains are highly cytolytic in vitro whereas others show virus replication with no apparent islet destruction. The capability to induce islet destruction is determined only partially by the virus serotype, since strain specific differences have been detected within some serotypes including echovirus 9 (E-9). In this study, the viral genetic factors determining the outcome of islet infection (i.e., destructive vs. benign) were investigated by constructing parallel infectious clones of lytic E-9-DM strain that was isolated from a small child at the clinical onset of type 1 diabetes. The capabilities of these clone-derived viruses to induce islet destruction were monitored and the lytic potential of clones was modified by site-directed mutagenesis. The lytic capabilities of these clone-derived viruses in human pancreatic islets were modified by a single amino acid substitution (T81A) in the capsid protein VP1. The data presented outline the importance of amino acid point mutations in the pathogenetic process leading to islet necrosis. However, although the amino acid substitution (T81A) modifies the lytic capabilities of E-9-DM strain-derived microvariant strains, it is likely that additional viral genetic determinants of pancreatic islet pathogenicity exist in other E-9 strains.

Buthionine sulfoximine inhibits cytopathic effect and apoptosis induced by infection with human echovirus 9.

We studied the effect of buthionine sulfoximine (BSO) on the replication of an isolate of human echovirus 9 (EV9) and the apoptosis induced by it in GMK cells. One hundred microM BSO markedly inhibited the cytopathic effect (CPE) induced by EV9. BSO also significantly inhibited apoptosis induced by EV9. BSO did not influence replication of EV9 genome, but inhibited virion formation. These results suggest that the inhibition by BSO of CPE and apoptosis induced by EV9 may be associated with the impairment of virion formation. Moreover, apoptosis induced by infections of human poliovirus 3, human coxsackievirus B5, A10 and A16, which, like EV9, belong to the genus Enterovirus, was markedly abolished by BSO. This finding suggests that enteroviral infections cause apoptosis through the activation of a common pathway that can be inhibited by BSO.

Echovirus-9 protein 2C binds single-stranded RNA unspecifically.

Polypeptide 2C is essential for picornavirus replication. Although many data on multiple functions of this highly conserved protein are available, the mechanism of RNA binding is still obscure. In this work, protein 2C of echovirus-9 strain Barty was expressed as a histidine-tagged protein in E. coli followed by nondenaturing purification to homogeneity. After incubation of 2C protein with different kinds of RNA fragments, binding was shown in gel retardation assays. Competition experiments revealed that 2C targets linear RNA unspecifically; however, single-stranded linear DNA does not react with this protein. In contrast to poliovirus, protein 2C of echovirus-9 only recognizes RNA with a low content of secondary structures. This may be a first hint of a different binding specificity of 2C in echo- and polioviruses.

Integrin alpha(v)beta3 (vitronectin receptor) is a candidate receptor for the virulent echovirus 9 strain Barty.

The enterovirus echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the alpha(v)beta3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this echovirus infection is controlled by the availability of alpha(v)beta3 integrin.

Cell attachment and mouse virulence of echovirus 9 correlate with an RGD motif in the capsid protein VP1.

The recently analyzed sequences of the nonpathogenic prototype strain Hill and the mouse-virulent strain Barty of the human echovirus 9 differ particularly in an insertion coding for an RGD motif at the C-terminus of the capsid protein VP1 in the genome of strain Barty. To investigate molecular determinants of virulence, we generated a panel of recombinant viruses derived from cDNA clones of strains Hill and Barty. In this communication, we show that the mouse-pathogenic character of strain Barty correlates with a 310-aa segment including the RGD motif. By mutating the RGD to an RGE tripeptide, the infectivity of the resulting echovirus 9 clones for GMK cells is lost. Furthermore, we could show that synthetic peptides containing the RGD sequence influence binding of mouse-virulent echovirus 9 strains to GMK cells, whereas binding of apathogenic strains is not affected. These results suggest that the RGD motif is a significant factor affecting pathogenicity of echovirus 9 strains.

SDZ 35-682, a new picornavirus capsid-binding agent with potent antiviral activity.

SDZ 35-682 is a potent and selective inhibitor of the replication of members of the picornavirus group. It inhibits several rhinovirus serotypes and echovirus 9 at concentrations as low as 0.1 micrograms/ml, without exerting any effect on cell proliferation up to 30 micrograms/ml. As observed with other capsid-binding antipicornavirus compounds, there is a wide variation in sensitivity of the different serotypes within the rhinovirus group. The point of interference of SDZ 35-682 in a single cycle of virus growth is an early event taking place before 2 or 3 h of echo- or rhinovirus replication, respectively. By incorporation of neutral red into the viral capsid and measurement of acquisition of photoresistance it is shown that uncoating of echovirus 9 is inhibited by SDZ 35-682. In addition, efficiency of adsorption of echovirus 9 is reduced by SDZ 35-682. To demonstrate that SDZ 35-682, like other uncoating inhibitors of picornaviruses, binds to the hydrophobic pocket beneath the canyon floor co-crystallization with HRV 14 was performed. Considerable conformational changes occur in VP1 in the HRV 14/SDZ 35-682 complex. SDZ 35-682 is 19 A long from end to end and thus fills the entire hydrophobic pocket including its innermost end; it is less flexible than other long antiviral agents. It has been suggested that compounds filling the entire hydrophobic pocket will affect the uncoating process of the virion. Thus, inhibition of viral uncoating, as demonstrated with echovirus 9, probably is the predominant mode of action of SDZ 35-682.

Acid stability of hepatitis A virus.

The acid stability of unpurified and highly purified hepatitis A virus (HAV) was tested and compared with that of poliovirus type 1, coxsackievirus types A9 and B1 and echovirus type 9. Only HAV had a high residual infectivity after 2 h of exposure to pH 1 at room temperature, remaining infectious for up to 5 h. At 38 degrees C, pH 1, HAV remained infectious for 90 min. Highly purified HAV was found to be infectious for 8 h at pH 1 and room temperature. This indicates that the increased stability is not due to protection by cellular material attached to the virus, but is a virus-specific marker. Under the same conditions, at pH 1 and room temperature, unpurified and highly purified HAV antigens were traceable for 5 and 4 h respectively.

Interaction between enteroviruses and human endothelial cells in vitro. Alterations in the physical properties of endothelial cell plasma membrane and adhesion of human granulocytes.

Fluorescent molecular probes were used for study of the interaction between enteroviruses (Echo 9, Echo 12, and Coxsackie B3 virus) and human endothelial cells in monolayer culture. With the use of the monomer-excimer method with pyrene decanoic acid it was shown that a marked dose-dependent restructuring of the plasma membrane occurred following addition of virus to the endothelial cells. This took the form of an increase in the lipid surface available to the lipophil reporter molecules, probably due to an alteration in the domain structure of the plasma membrane caused by insertion of virus capsid proteins. Experiments with diphenylhexatriene indicated that the enteroviruses had only a slight tendency to make the plasma membrane of the endothelial cell more fluid. Concomitant with these alterations in the biophysical properties of the membrane, a virus-induced increase in granulocyte adherence to the endothelial cells was observed for all three enteroviruses studied. Possible mechanisms for this elevated adherence are discussed, as well as the significance of the results for the phenomenon of virus-induced granulocytopenia.

F-Met-Leu-Phe and echo 9 virus interaction with human granulocytes. Changes of cell membrane structure.

Biophysical and biochemical methods were applied for investigation of cell membrane properties of human polymorphonuclear leukocytes (PMNs) exposed to the chemotactic peptide N-formylmethionyl-leucylphenylalanine (f-Met-Leu-Phe) and echovirus type 9, strain A, Barty. Steady-state fluorescence depolarization with diphenylhexatriene demonstrated no gross changes of the total membrane fluidity under the different experimental conditions. However, by means of the monomer-excimer technique with pyrenedecanoic acid (PDA), significant changes of the local membrane structure were detected for both agents. As demonstrated by a higher excimer ratio, the membrane area available for the PDA molecules was restricted by f-Met-Leu-Phe. This effect was dependent on the dose and on the time of interaction of the chemotactic peptide. These experimental findings were explained by the formation of functional receptor units ("activated membrane"). Echo 9 virus exhibited the opposite effect, characterized by a higher ratio of monomers, which also depended on the viral dose and the time of virus-PMN interaction. These virus-induced findings were explained by the dissolution of functional receptor units. Consecutive exposure of the PMNs to f-Met-Leu-Phe and echovirus, or vice versa, demonstrated a virus-predominant effect on the membrane structures.

Echo 9 virus-induced order-disorder transition of chemotactic response of human polymorphonuclear leucocytes: phenomenology and molecular biology.

By means of functional, morphological, and biophysical methods the in vitro interaction of Echo virus, type 9, strain A. Barty with human polymorphonuclear leucocytes (PMNs) was investigated and analyzed by statistical methods. Control cells and virus-treated PMNs (15 min, 37 degrees C; PMN: virus (pfu)-ratio ranging from 1:1 to 1:50) were exposed to a chemotactic gradient (N-formylmethionyl-leucylphenylalanine = f-Met-Leu-Phe, 10(-8) M/mm) in a Zigmond chamber. Whereas the track velocity of the moving PMNs was not affected by the virus, the degree of orientation of virus-treated PMNs declined in a way dependent on the viral dose and on the time of PMN:virus interaction, resulting in a shift from chemotactic to chemokinetic response. This virus-induced order-disorder transition of chemotactic response can be described by a logarithmic law in analogy to the Weber-Fechner law. Parallel to the functional disturbances, virus-induced changes of cell shape, which could be confirmed by additional light and electron microscopy techniques, were also detected using statistical analysis of cytological data (median cell size, anisotropy of cell shape) by means of two-dimensional histograms. To investigate f-Met-Leu-Phe- or/and Echo 9 virus-induced PMN-cell membrane changes, the monomer-excimer technique with pyrenedecanoic acid as fluorescent probe was applied, which gives information about structural changes of the cell membrane. Addition of the chemotactic peptide (10(-8) M) to control PMNs resulted in a higher rate of excimer formation obviously due to the formation of new functional (receptor) units (= activated cell membrane). Echo 9 virus exhibited an opposite effect. Quantitative analysis of these results revealed that the f-Met-Leu-Phe-induced cell membrane changes were extinguished by the addition of 2 pfu Echo 9 virus. So far, we have additional indicators of a virus-induced order-disorder transition of chemotactic response of human PMNs on a molecular biological level.