Viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in ICAM-1-negative cells - PubMed (original) (raw)

Viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in ICAM-1-negative cells

A Reischl et al. J Virol. 2001 Oct.

Abstract

Major receptor group common cold virus HRV89 was adapted to grow in HEp-2 cells, which are permissive for minor group human rhinoviruses (HRVs) but which only marginally support growth of major-group viruses. After 32 blind passages in these cells, each alternating with boosts of the recovered virus in HeLa cells, HRV89 acquired the capacity to effectively replicate in HEp-2 cells, attaining virus titers comparable to those in HeLa cells although no cytopathic effect was observed. Several clones were isolated and shown to replicate in HeLa cells whose ICAM-1 was blocked with monoclonal antibody R6.5 and in COS-7 cells, which are devoid of ICAM-1. Blocking experiments with recombinant very-low-density lipoprotein receptor fragments and enzyme-linked immunosorbent assays indicated that the mutants bound a receptor different from that used by minor-group viruses. Determination of the genomic RNA sequence encoding the capsid protein region revealed no changes in amino acid residues at positions equivalent to those involved in the interaction of HRV14 or HRV16 with ICAM-1. One mutation was within the footprint of a very-low-density lipoprotein receptor fragment bound to minor-group virus HRV2. Since ICAM-1 not only functions as a vehicle for cell entry but has also a "catalytic" function in uncoating, the use of other receptors must have important consequences for the entry pathway and demonstrates the plasticity of these viruses.

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Figures

FIG. 1

HRV89 variants recovered after 32 adaptation cycles in HEp-2 cells are serotypically identical to wt virus. HEp-2 cell-adapted virus and wt virus were incubated for 90 min at 34°C with serial twofold dilutions (from left to right) of the serotype-specific antisera indicated. The mixtures were then transferred onto HeLa cell monolayers in 96-well plates. Tissue damage was monitored after 2 days at 34°C by crystal violet staining.

FIG. 2

Adaptation to growth in HEp-2 cells alters the sensitivity of HRV89 to ICAM-1. Virus was incubated with twofold serial dilutions of soluble ICAM-1 (left to right, starting with 25 μg/ml) for 90 min at 34°C. HeLa cells in 96-well plates were then challenged with the mixtures, and cytopathic effect was revealed by staining with crystal violet after 3 days. Numbers refer to individual clones.

FIG. 3

Stereo image of a ribbon model of VP1, VP2, and VP3 (blue, green, and red, respectively) of HRV89 as calculated with Swiss-Model. The positions of the mutations are represented in space-filling mode. Red, mutations identical in all three clones; blue, mutations present only in individual clones (compare to Table 5). (Top) View down the z axis; (bottom) view perpendicular to the z axis. Only some of the mutations are labeled. The fivefold axis of icosahedral symmetry is indicated. The canyon is clearly visible between 1087D and 1272S. Note that the loop between βF and βG in VP3 could not be modeled and thus is not closed. The figure was made with Swiss-Pdb-Viewer, version 3.5b1.

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Viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in ICAM-1-negative cells - PubMed (original) (raw)