Despite high conservation of pocket amino acids, polymorphism was detected at positions 1092, 1094 and 1180. Neither Pro-1094-->Thr nor Val-1180-->Ile altered efficacy of pleconaril treatment. But the amino acid at position 1092 was strongly associated with susceptibility of CVB3 to the capsid inhibitor. Whereas leucine was involved in resistance, isoleucine and valine were detected in pleconaril-susceptible CVB3. Results from antiviral assays with hybrid viruses demonstrate the crucial role of amino acid 1092 in pleconaril susceptibility. A resistant cDNA-generated CVB3 became pleconaril-susceptible after accepting parts from the genome region encoding Ile-1092 into its capsid. Computational analysis suggests that conformational changes in the hydrophobic pocket occur when leucine is substituted for isoleucine or valine and that this change leads to susceptibility to pleconaril.

Hammerschmidt E, Loeffler I, Wolf G. Morg1 heterozygous mice are protected from acute renal ischemia-reperfusion injury.

Group B coxsackieviruses (CVB) belonging to the family of nonenveloped picornaviruses utilize the coxsackievirus-adenovirus receptor (CAR) to bind to and enter into host cells (5). Small depressions surrounding the fivefold axis, the so-called canyons formed by the viral capsid proteins VP1, VP2, and VP3, bind the CAR (24). Receptor binding induces conformational changes which facilitate the internalization of viral RNA into host cells (18,26). Additionally, human decay accelerating factor (hDAF/CD55) functions as an attachment but not an entry receptor for CVB1, -3, and -5 (6, 32). Recently, cell surface heparan sulfate proteoglycans (HSPG) were identified as additional receptors for the CVB3 variant PD (CVB PD) (38). Using HSPG for infection, CVB3 PD also replicates in CAR-lacking cell lines, e.g., CHO-K1, BHK-21, RD, and L929 (29).HSPG consist of a polydisaccharide chain tethered to serine residues of defined core proteins by a linking tetrasaccharide composed of xylose-galactose-galactose-glucuronic acid (12). NAcetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA) residues are added alternatingly to the linker tetrasaccharide. Several steps of modifications of GlcNAc and GlcA then follow, including N-deacetylation and N-sulfation of GlcNAc, C-5 epimerization of GlcA to L-idoronic acid (IdoA), 2-O-sulfation of the uronic acid, and 6-O-and 3-O-sulfation of D-glucosamine (GlcN) residues. The resulting high molecular diversity of heparan sulfate (HS) chains enables many specific interactions with very different proteins and glycoproteins, e.g., growth factors, cytokines, and human pathogens, including enveloped viruses (13,22,36). Cell surface HS were also shown to bind nonenveloped viruses, e.g., a variant of human rhinovirus 89 (HRV89) (37), echoviruses (15), swine vesicular disease virus (11), and Theiler's murine encephalomyelitis virus (25), belonging, like CVB3 PD, to the picornavirus family. The sulfated structural motifs of HS mediating binding or entry of picornaviruses are poorly known. Strong differences in viral replication in CHO cell mutants with different defects in heparan sulfate synthesis led to the assumption that CVB3 PD binds to specifically sulfated HS moieties.During this study, the following tasks were performed: (i) the structural requirements, especially the sulfation pattern of HS necessary for CVB3 PD entry, were examined by using competition assays with growth factors binding to specifically sulfated HS sequences as well as with specifically desulfated heparins; (ii) the entry pathway of CVB3 PD while using HS was studied; and (iii) the kinetics of viral entry and the viral life cycle depending on the presence of CAR or HS as the receptor were investigated. MATERIALS AND METHODSCell lines and viruses. Chinese hamster ovary cells (CHO-K1; German Collection of Microorganism and Cell Cultures no. ACC-110) and heparan sulfatenegative, human CAR-transfected pgsD-677-hCAR cells (38) were grown in Dulbecco's modified Eagle's medium (Cambrex, Belgium) supplemented with 10% fetal bovine...

The reasons for the different outcome of coxsackievirus B3 (CVB3)-induced heart disease in humans are not well understood. Since there are no experimental data on the course of disease after infection with genetically different CVB3 in a natural variable population until now, we studied the outcome of virus infection in outbred NMRI mice after inoculation of genetically different CVB3 variants. Adult male mice were inoculated with seven closely related CVB3 variants. The histopathological changes of heart and pancreas tissue, antibody induction, virus titers, and persistence of viral positive- as well as negative-strand RNA in spleen and heart tissue were compared at day 7 or day 28 after infection to detect prerequisites and predictive factors for chronic myocarditis. Six CVB3 variants infected NMRI mice. CVB3 infection (i) did not induce detectable myocardial injury, (ii) caused signs of healing up acute myocarditis or (iii) ongoing chronic myocarditis. Neither IgG antibody responses nor the extent of destruction of exocrine pancreatic tissue or viral RNA load in spleen did correlate with myocardial histopathology. In contrast, a high persistent viral RNA load in heart tissue specimens was characteristic for mice developing chronic myocarditis. The results of the present study corroborate high viral load in the acute stage of myocarditis and high amounts of persisting CVB3 RNA in heart tissue as predictive marker of chronic myocarditis. The outcome of CVB3-induced heart disease in outbred NMRI mice depends strongly on the viral genetic background. In particular an important role of viral capsid proteins is suggested.