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Comparative Study
. 2010 Oct;84(19):9695-708.
doi: 10.1128/JVI.00071-10. Epub 2010 Jul 14.

Characterization of a putative ancestor of coxsackievirus B5

Maria Gullberg  1 , Conny Tolf, Nina Jonsson, Mick N Mulders, Carita Savolainen-Kopra, Tapani Hovi, Marc Van Ranst, Philippe Lemey, Susan Hafenstein, A Michael Lindberg
Affiliations
Comparative Study

Characterization of a putative ancestor of coxsackievirus B5

Maria Gullberg et al. J Virol. 2010 Oct.

Abstract

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.

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Figures

FIG. 1.
FIG. 1.
Genomic structures of CVB5D and CVB5-P1anc. An illustration of the genome organization of CVB5 is shown at the top, including positions of relevant restriction enzyme sites used to construct infectious viral cDNA clones. The ClaI site (*) was introduced to construct a cassette vector. Infectious CVB5D cDNA clone variants, as they were inserted into the pCR-Script Direct SK(+) vector, are depicted at the bottom. The ancestral P1 sequence is indicated in gray. Names of constructed cDNA clones and viruses generated from these clones (given within parentheses) are used throughout the text. UTR, untranslated region.
FIG. 2.
FIG. 2.
Phylogenetic relationships among CVB5 isolates. (A) Phylogram based on the nucleotide sequences of the VP1 gene. •, CVB5 isolates selected for sequencing of the entire P1 region. (B) Phylogeny of selected CVB5 isolates based on the nucleotide sequences of the P1 region. The scale bars represent the genetic distance (nucleotide substitutions per site). Both ML trees were evaluated by nonparametric bootstrap analysis and 1,000 pseudoreplicates. Only bootstrap values ≥70% are denoted. Clusters are indicated by roman numerals (clusters I and II). The trees were rooted with CVB4T and CVB6S. Abbreviations in isolate names are as follows: den, Denmark; dor, Dominican Republic; ecu, Ecuador; est, Estonia; fin, Finland; fra, France; hon, Honduras; jap, Japan; kyr, Kyrgyzstan; net, Netherlands; pak, Pakistan; rom, Romania; rus, Russia; spa, Spain; uk, United Kingdom; usa, United States of America. The last two numbers of isolates depict the year of isolation, except for isolates from France, where the year of isolation is shown by the two first numbers after fra.
FIG. 3.
FIG. 3.
Root-to-tip divergence plot. Shown is a linear regression plot for root-to-tip divergence versus sampling year. The SVDV isolates included in the analysis are encircled.
FIG. 4.
FIG. 4.
Maximum clade credibility tree representing the CVB5 evolutionary history inferred by using Bayesian evolutionary analysis. The tree has branch lengths in time units and is depicted on a time scale. The uncertainty (95% highest posterior density intervals) for the node times is indicated with blue bars. Branches with an asterisk are supported with posterior probabilities higher than 0.85. Rate variation among branches is indicated by using a blue-black-red (slow-average-high) color scheme. Clusters are indicated by roman numerals (clusters I and II).
FIG. 5.
FIG. 5.
Structural differences between CVB5-P1anc and clinical CVB5 isolates. (A, left) A CVB3 (PDB accession number 1COV) (64) protomer in a ribbon diagram with VP1, VP2, VP3, and VP4 (light blue, light green, pink, and light yellow, respectively) with a symmetry-related copy of VP3 included to complete the canyon. Based on a ClustalW alignment of CVB5-P1anc and the CVB5 isolates with CVB3, the sequence-equivalent residues that differ between CVB5-P1anc and the CVB5 isolates are depicted as spheres, and VP1, VP2, VP3, and VP4 are shown in dark blue, green, red, and yellow, respectively. The asymmetric unit is indicated by a black triangle. (Right) A single pentamer of the virus capsid is surface rendered to show the location of the amino acid differences exposed to the viral surface. (B) Surface-rendered close-up of the pocket with VP1 residues 93 and 178 (i.e., residues 95 and 180 of CVB5) that line the pocket. The pocket factor is shown in orange (64). On the right is the pentamer showing the amino acid differences exposed to the interior surface of the capsid. (C) Residues predicted to interact with CAR and DAF. (Left) The protomer is shown in a ribbon diagram, with residues within the CAR and DAF footprints shown as magenta and cyan spheres (36, 39). A symmetry-related copy of VP3 is shown to provide the entire CAR footprint on CVB3 in one asymmetric unit. (Right) In the surface-rendered pentamer, the CAR footprint on CVB3 is in magenta, and the DAF binding footprint is in cyan.
FIG. 6.
FIG. 6.
Viral titers of cDNA clone-derived viruses. The titers were determined at 5 days after transfection of cDNA clones into HeLa cells by endpoint titration. Values shown are means ± SEM (n = 3).
FIG. 7.
FIG. 7.
CVB5-P1anc infection in HeLa cells. (A) Light microscopic image of CVB5-P1anc-infected (MOI of 10) HeLa cells (12 h p.i.). Bar, 100 μm. (B) Production of viral antigen in HeLa cells infected with CVB5-P1anc (MOI of 10) and analyzed at 5 h after infection. Antigen was detected with an enterovirus-specific polyclonal rabbit antibody (KTL-482) and a secondary antibody labeled with Alexa Fluor 488 (green). The cellular nuclei were visualized with DAPI (blue). Bar, 50 μm.
FIG. 8.
FIG. 8.
Flow cytometric analysis of CAR and DAF expression on CHO, CHO-CAR, CHO-DAF, and HeLa cells. For the detection of CAR and DAF, monoclonal anti-CAR (RmcB) and anti-DAF (BRIC110) antibodies were used (black histograms). A mouse IgG1 antibody was used as a negative control (gray histograms).
FIG. 9.
FIG. 9.
Receptor preferences, plaque phenotypes, and virus growth kinetics of CVB5-P1anc, CVB5Dwt, 151rom70, and 4378fin88 as well as CVB5-P1anc infectivity in different cell lines. (A) Binding of radiolabeled CVB5 viruses to CHO, CHO-CAR, CHO-DAF, or HeLa cells. Cells were incubated with [35S]methionine-cysteine-labeled CVB5-P1anc, CVB5Dwt, 151rom70, or 4378fin88 at room temperature for 2 h. Following the removal of unbound virions, the cell-associated radioactivity was determined by scintillation counting. Results are presented as means ± SEM (n = 3). (B) Plaques were visualized at 48 h p.i. by crystal violet staining of HeLa cells. Virus-infected cell lysates were diluted 10−7 times in order to distinguish individual plaques. (C) HeLa cells were infected with the indicated viruses at an MOI of 10 TCID50/cell. At various times postinfection, samples were frozen, and the total yield of infectious virus was quantified by the TCID50 method. Results shown are representative of three independent experiments. (D) CVB5-P1anc titer determined at time point zero and after complete CPE or 5 days p.i. by endpoint titration in HeLa cells. Results are presented as means ± SEM (n = 3).
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References

    1. Adey, N. B., T. O. Tollefsbol, A. B. Sparks, M. H. Edgell, and C. A. Hutchison III. 1994. Molecular resurrection of an extinct ancestral promoter for mouse L1. Proc. Natl. Acad. Sci. U. S. A. 91:1569-1573. - PMC - PubMed
    1. Arnberg, N., K. Edlund, A. H. Kidd, and G. Wadell. 2000. Adenovirus type 37 uses sialic acid as a cellular receptor. J. Virol. 74:42-48. - PMC - PubMed
    1. Baranowski, E., N. Sevilla, N. Verdaguer, C. M. Ruiz-Jarabo, E. Beck, and E. Domingo. 1998. Multiple virulence determinants of foot-and-mouth disease virus in cell culture. J. Virol. 72:6362-6372. - PMC - PubMed
    1. Bedard, K. M., and B. L. Semler. 2004. Regulation of picornavirus gene expression. Microbes Infect. 6:702-713. - PubMed
    1. Bergelson, J. M., J. A. Cunningham, G. Droguett, E. A. Kurt-Jones, A. Krithivas, J. S. Hong, M. S. Horwitz, R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-1323. - PubMed

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