An antiviral mechanism of nitric oxide: inhibition of a viral protease.
Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses. (+info)
Inhibition of Echovirus-12 multiplication by N-carbobenzoxy-D-glucosamine.
The glucosamine derivative, N-carbobenzoxy-D-glucosamine (NCBZG) inhibits the multiplication of Echovirus-12 and the synthesis of both virus RNA and protein at a stage in the virus growth cycle after attachment and penetration. However, the compound does not inhibit virus multiplication after the appearance of progeny virus nor after virus RNA has accumulated. Incorporation of radioactive glucosamine and choline into infected and uninfected cultures is inhibited by NCBZG as is the virus-induced increase in choline incorporation. The compound also prevents the appearance of radioactive choline in isolated membranous structures. The compound did not alter significantly the cellular RNA or protein synthesis, plating efficiency of the cells, their growth over a period of several days, nor the virus-directed inhibition of cellular RNA and protein. These findings suggest that the compound inhibits virus multiplication by its effect on the initiation of biosynthesis which appears to require membrane synthesis. (+info)
A new picornavirus isolated from bank voles (Clethrionomys glareolus).
A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype. (+info)
Identification of neutralizing epitopes on a European strain of swine vesicular disease virus.
Six neutralizing monoclonal antibodies (MAbs) were used to isolate MAb neutralization-resistant (MAR) mutants from a recent European strain of swine vesicular disease virus (SVDV), ITL/9/93. Sequencing of MAR mutants identified two epitopes located at positions analogous to sites 2A (VP2) and 3B (VP3) on poliovirus (PV) which have been previously identified on a Japanese strain of SVDV. A third epitope near to the C terminus of VP1, not previously recognized on SVDV, was tentatively identified in a region analogous to site 1 of PV. A fourth epitope, located in the C-terminal region of VP3, has never before been recognized as a site of neutralization on picornaviruses. All four epitopes were predicted to be surface-exposed. (+info)
Serum albumin inhibits echovirus 7 uncoating.
Echoviruses induce a wide spectrum of diseases in man, the most severe being meningitis. In neonates, however, a severe systemic infection can be observed, leading to death. Serum albumin is the most abundant protein in plasma and most interstitial fluids, and its functions include osmoregulation and transport and delivery of hydrophobic molecules such as fatty acids and steroids. The results of cold-synchronized one-step growth analysis of echovirus 7 infection and sucrose-gradient analysis of A-particles suggest that physiological concentrations of albumin block echovirus 7 infection by inhibiting uncoating. The blockage was reversible and was still effective when albumin was added 30 min after virus adsorption. Inhibition of uncoating was confirmed by using rhodanine, a known specific inhibitor of echovirus uncoating. After removal of the albumin blockage, addition of rhodanine perpetuated the inhibition. Serum and interstitial albumin concentrations may limit echovirus infection in vivo and thereby act as an extracellular determinant for echovirus tropism. (+info)
Molecular evolution of swine vesicular disease virus.
Phylogenetic analysis was used to examine the evolutionary relationships within a group of coxsackie B viruses that contained representatives of the major serotypes of this group and 45 isolates of swine vesicular disease virus (SVDV) from Asia and Europe. Separate analyses of sequence data from two regions of the viral genomes encoding the VP1 and 3BC genes both revealed that the SVDV belonged to a single monophyletic group which could be clearly distinguished from all other sampled coxsackieviruses. Regression analysis revealed that within the SVDV clade at least 80% of the synonymous variation in evolutionary divergence between isolates was explained by time, indicating the existence of an approximate molecular clock. Calibration of this clock according to synonymous substitutions per year indicated the date of occurrence of a common ancestor for the SVDV clade to be between 1945 and 1965. (+info)
Viral myocarditis: identification of five differentially expressed genes in coxsackievirus B3-infected mouse heart.
Differences in host susceptibility to viral myocarditis caused by a given strain of coxsackievirus B3 (CVB3) are known to be largely related to host genetic factors. Little is known, however, about the key genes that encode determinants (mediators) of myocarditis development or the nature of injury. To identify these genes and further understand the molecular mechanisms of the disease process, we have used a murine model and the differential display technique to fingerprint mRNAs from CVB3-infected mouse hearts. Total RNA was extracted from hearts of 4- and 10-week-old A/J(H-2(a)) mice at day 4 after CVB3 infection, and mRNAs were detected by reverse transcriptase-polymerase chain reaction and subsequently analyzed on polyacrylamide DNA sequencing gels. The differentially displayed bands were confirmed by Northern hybridization using the bands as cDNA probes. Twenty-eight upregulated or downregulated bands were selected from the sequencing gels; among these, 2 upregulated and 3 downregulated cDNA fragments were confirmed by Northern hybridization. DNA sequence analysis and GenBank searching have determined that 4 of the 5 candidate genes are homologous to genes encoding Mus musculus inducible GTPase, mouse mitochondrial hydrophobic peptide (a subunit of NADH dehydrogenase), mouse beta-globin, and Homo sapiens cAMP-regulated response element binding protein (CREB) binding protein (CBP), respectively. The remaining candidate gene matches an unpublished cDNA clone, M musculus Nip21 mRNA (GenBank accession number, AF035207), which is homologous to human Nip2, a Bcl-2 binding protein. Our data suggest preliminarily that both structural and nonstructural genes are involved in myocarditis development. For the structural gene, beta-globin, we further confirmed its downregulation at the protein level by measuring the mean cell volume of red blood cells and found it was marginally reduced in the CVB3-infected group (P<0.06), with no change in hemoglobin concentration. Cardiac myoglobin concentration was also measured and found to be decreased (P<0.005), with a parallel decrease in total soluble protein in the CVB3-infected mouse myocardium (P<0.01). We also noted that the ratio of myoglobin to total protein was not significantly changed; this may be due to the downregulation of additional genes in the host heart, a number being observed on the differential display gels. The significant downregulation of beta-globin major gene expression in the heart may be relevant to impaired cardiac function in both the early and late postinfection period. The other identified nonstructural genes are known to be involved in regulation of gene expression, signal transduction pathways, and apoptotic cell death. The altered expression of structural and nonstructural genes may play important roles in the mediation of myocarditis development and perhaps other pathological processes in the heart. (+info)
Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication.
Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks. (+info)