Previous work from our laboratory demonstrated that selenium deficiency in the mouse allows a normally benign (amyocarditic) cloned and sequenced Coxackievirus to cause significant heart damage. Furthermore, Coxsackievirus recovered from the hearts of selenium-deficient mice inoculated into selenium-adequate mice still induced significant heart damage, suggesting that the amyocarditic Coxsackievirus had mutated to a virulent phenotype. Here we report that sequence analysis revealed six nucleotide changes between the virulent virus recovered from the selenium-deficient host and the avirulent input virus. These nucleotide changes are consistent with known differences in base composition between virulent and avirulent strains of Coxsackievirus. To the best of our knowledge, this is the first report of a specific nutritional deficiency driving changes in a viral genome, permitting an avirulent virus to acquire virulence due to genetic mutation.

Rapid genomic evolution of a non-virulent coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates.

Digital-imaging microscopy was performed to study the effect of Coxsackie B3 virus infection on the cytosolic free Ca2+ concentration and the Ca2+ content of the endoplasmic reticulum (ER). During the course of infection a gradual increase in the cytosolic free Ca2+ concentration was observed, due to the influx of extracellular Ca2+. The Ca2+ content of the ER decreased in time with kinetics inversely proportional to those of viral protein synthesis. Individual expression of protein 2B was sufficient to induce the influx of extracellular Ca2+ and to release Ca2+ from ER stores. Analysis of mutant 2B proteins showed that both a cationic amphipathic alpha-helix and a second hydrophobic domain in 2B were required for these activities. Consistent with a presumed ability of protein 2B to increase membrane permeability, viruses carrying a mutant 2B protein exhibited a defect in virus release. We propose that 2B gradually enhances membrane permeability, thereby disrupting the intracellular Ca2+ homeostasis and ultimately causing the membrane lesions that allow release of virus progeny.

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Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release

The structure of coxsackievirus B3 at 3.5 å resolution

Coxsackievirus B3 (CVB3) infections induce myocarditis in humans and mice. Little is known about the molecular characteristics of CVB3 that activate the cellular immunity responsible for cardiac inflammation. Previous experiments have identified an antibody escape mutant (H310A1) of a myocarditic variant of CVB3 (H3) that attenuates the myocarditic potential of the virus in mice in spite of ongoing viral replication in the heart. We have cloned full-length infectious cDNA copies of the viral genome of both the wild-type myocarditic H3 variant of CVB3 and the antibody escape mutant H310A1. Progeny viruses maintained the myocarditic and attenuated myocarditic potential of the parent viruses, H3 and H310A1. The full sequence of the H3 viral cDNA is reported and compared with those of previously published CVB3 variants. Comparison of the full sequences of H3 and H310A1 viruses identified a single nonconserved mutation (A to G) in the P1 polyprotein region at nucleotide 1442 resulting in an asparagine-to-aspartate mutation in amino acid 165 of VP2. This mutation is in a region that corresponds to the puff region of VP2. Nucleotide 1442 of the H3 and H310A1 cDNA copies of the viral genome was mutated to change amino acid 165 of VP2 to aspartate and asparagine, respectively. The presence of asparagine at amino acid 165 of VP2 is associated with the myocarditic phenotype, while an aspartate at the same site reduces the myocarditic potential of the virus. In addition, high-level production of tumor necrosis factor alpha by infected BALB/c monocytes is associated with asparagine at amino acid 165 of VP2 as has been previously demonstrated for the H3 virus. These findings identify potentially important differences between the H3 variant of CVB3 and other previously published CVB3 variants. In addition, the data demonstrate that a point mutation in the puff region of VP2 can markedly alter the ability of CVB3 to induce myocarditis in mice and tumor necrosis factor alpha secretion from infected BALB/c monocytes.

A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3.

IP: 54.70.40.11 On: Sat, 08 Dec 2018 03:07:20 Journal of General Virology (1997), 78, 1–11. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Classification of enteroviruses based on molecular and biological properties.

A full-length reverse-transcribed, infectious cDNA copy of coxsackievirus B3 (CVB3) was used to determine the nucleotide sequence of this cardiotropic enterovirus. Comparison of the nucleotide sequence and the deduced amino acid sequence of the viral precursor polyprotein with the sequences of other group B coxsackieviruses (CVB1 and CVB4) demonstrates a high degree of genetic identity. They share about 80% homology at the nucleotide level and about 90% when the amino acid sequences of the polyproteins are compared. The potential processing sites of the coxsackievirus polyproteins, as deduced from alignment with the poliovirus sequence, are conserved among these enteroviruses with the exception of the cleavage sites between VP1 and 2Apro and between polypeptides 2B and 2C. Comparison of the 5' termini of the enteroviral genomes reveals a high degree of identity, including the initial 5' consensus UUAAAACAGC, suggesting essential functions in virus replication. An important finding concerning the molecular basis of infectivity was that both recombinant CVB3 cDNA and in vitro-synthesized CVB3 RNA transcripts are infectious, although two initial 5' uridine residues found on the authentic CVB3 RNA were missing. Here, we report that cDNA-generated CVB3, as well as CVB3 generated by in vitro-synthesized RNA transcripts, regains the authentic initial 5' uridine residues during replication in transfected cells, indicating that the picornaviral primer molecule VPg-pUpU may be uridylylated in a template-independent fashion. The generation of virus or virus mutants with infectious recombinant CVB3 cDNA and in vitro-synthesized infectious CVB3 transcripts should provide a valuable means for studying the molecular basis of the pathogenicity of this cardiotropic enterovirus.

I Bergmann

Papers

Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA: two initial 5' uridine residues are regained during plus-strand RNA synthesis.