Birgit Nelsen-Salz

Molecular cloning and sequence determination of the complete genome of the virulent echovirus 9 strain Barty

As part of a study of the molecular basis of pathogenicity of echovirus 9, the complete nucleotide sequence of the mouse-virulent echovirus 9 strain Barty was determined. Excluding the poly(A) tail, the complete RNA genome is composed of 7451 bases. The postulated open reading frame extends from nucleotide (nt) 741 to 7349 and predicts a polyprotein of 2203 amino acids (aa). As compared with the sequence of the echovirus 9 prototype strain Hill, which is apathogenic for newborn mice, 1492 nt are exchanged, leading to 9% divergence of the deduced amino acid sequence. The foremost difference between both strains is located at the C-terminus of the capsid protein VP1. In the case of strain Barty, an additional 10 aa fragment, including an RGD motif, is inserted.

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 αvβ3 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 αvβ3 integrin.

Analysis of sequence and pathogenic properties of two variants of encephalomyocarditis virus differing in a single amino acid in VP1.

The encephalomyocarditis (EMC) virus-induced diabetes-like syndrome in mouse inbred strains was used as a model to study the insulin-dependent diabetes mellitus (IDDM). Our investigations were performed with two EMC virus variants, PV2 and PV7. After infection of SJL mice with 10(5) PFU of PV2 about 70% of the animals developed a diabetes-like syndrome, whereas the PV7 infected mice appeared healthy. Histological examination and in situ experiments revealed that the islets of Langerhans are a main target of PV2, whereas PV7 infection leads to only modest changes of the islets. Sequence analysis of both variants revealed one amino acid exchange within the capsid protein VP1. Hence, we describe the first diabetogenic and non-diabetogenic EMCV variants differing in only one single amino acid.

The picornavirus replication inhibitors HBB and guanidine in the echovirus-9 system: the significance of viral protein 2C.

HBB [2-(alpha-hydroxybenzyl)-benzimidazole] and guanidine are potent inhibitors of picornavirus replication. Among other evidence, limited cross-resistance and a synergistic effect of both inhibitors suggest similar but not identical mechanisms of antiviral action. Echovirus-9 variants resistant to each of these drugs were characterized and sequenced. Complete resistance to HBB or guanidine was shown to be due to single but different point mutations in the non-structural protein 2C. Protein 2C was expressed as GST fusion and His-tagged proteins for the wild-type and various mutants. Although three mutations were located in or near conserved NTP binding motifs, NTPase activity was not altered in the presence of HBB or guanidine.

Echovirus 9 strain Barty non-structural protein 2C has NTPase activity

Non-structural protein 2C is known to play a fundamental role in the replication of picornaviruses. Sequence analyses revealed that 2C belongs to a rapidly expanding group of proteins containing a consensus sequence for nucleotide binding (NTB). We report that echovirus 9 polypeptide 2C displays NTPase activity in vitro. In our experiments, several P2 genes were expressed in Escherichia coli as fusion proteins linked to glutathione S-transferase (GST) prior to purification close to homogeneity. In contrast to GST-2B, both GST-2C and GST-2BC showed ATPase as well as GTPase activity indicating that the site for NTB binding and splitting is located in 2C.

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.

Determinants of pathogenicity of echovirus 9 in men: significance of a functional RGD-Motif

In this study, we investigated nine independent echovirus 9 isolates obtained from sick children in 1995. It is discovered that these isolates differ in respect to their pathogenicity for newborn mice indicating that the degree of human pathogenicity of an echovirus 9 variant does not necessarily correlate with mouse pathogenicity. Nevertheless, all virus variants are found to code for an RGD-motif within their VP1 protein. Hence, the RGD-motif and its highly conserved flanking regions are the conditio sine qua non, but, as expected, not sufficient for the mouse-pathogenic character.

HPLC is an effective and fast method for analysis of viral proteins : a study of encephalomyocarditis virus mutants differing in pathogenicity

We investigated the use of HPLC in analysis of picornavirus variants by comparing structural polypeptides of three stable mutants of encephalomyocarditis virus (EMCV). The variants are known to differ in their pathogenicity for mice: plaque variant 2 (PV2) is diabetogenic, PV7 is non-diabetogenic and PV21 induces a generalized lethal infection. We first used HPLC to separate the structural proteins at high purity levels. Detailed analysis of these structural proteins by HPLC-peptide mapping revealed differences in all four viral proteins of PV21 as compared with mutants PV2 and PV7. A single amino acid exchange was found in viral protein 1 between PV2 and PV7. Altered peaks were identified by calculating retention times of tryptic peptides using sequence data and a computer program. Since peak alterations could be attributed to the observed amino acid exchanges, the results correlate well with cDNA sequencing data. Thus HPLC proved to be a useful and fast tool for primary or additional characterization of picornavirus variants at the level of whole virus proteins.

Fatty Acid-Depleted Albumin Induces the Formation of Echovirus A Particles

ABSTRACT Picornavirus infection requires virus uncoating, associated with the production of 135S “A” particles and 80S empty particles from 160S mature virions, to release the RNA genome into the cell cytoplasm. Normal albumin inhibits this process. We now show that when depleted of fatty acids, albumin induces the formation of echovirus A particles.