Elizabeth Hewat

Institut de Biologie Structurale J-P Ebel, Grenoble, France

The attachment of a virus to specific cell-surface receptors is a key event in the life cycle of animal viruses. It determines the host range and tropism of infection and initiates delivery of the genome into the cell. Once bound to a receptor, the non-enveloped viruses such as the rhinoviruses must then transfer their genome directly across a membrane into the cytoplasm for reproduction [1].

Human rhinoviruses (HRVs) are a major cause of the common cold. They are small, icosahedral viruses, 300 A in diameter, and belong to the Picornaviridae family, which includes Rhinovirus, Aphthovirus, Enterovirus, Cardiovirus, etc. Their capsid is composed of 60 copies each of four viral coat proteins, VP1, VP2, VP3, and VP4, on a T = 1 (or pseudo T=3) icosahedral lattice [2]. The three major capsid proteins VP1, VP2, and VP3 all have the same basic eight-stranded P-barrel fold and a molecular weight of around 30 kDa. VP4 is a small protein located inside the capsid. The capsid encloses a single positive RNA strand of about 7000 bases. The HRV capsid has a star-shaped dome on each of the five-fold axes surrounded by a shallow depression or "canyon" and a triangular plateau centered on each three-fold axes and around each five-fold axes (Fig. 1).

A distinctive feature of VP1 is that it has a "pocket" or hollow within the P-barrel that is accessible from the exterior of the capsid. This hydrophobic pocket located at the base of the canyon is frequently occupied by a natural pocket factor, a fatty-acid-like molecule. This pocket factor is believed to stabilize the virus during its spread from cell to cell [3].

With one exception, HRVs are classified into a major group and a minor group based on their specificity for cell receptors (Fig. 2). The major group HRVs bind to the intercellular adhesion molecule-1 (ICAM-1) [4], which belongs to the immunoglobulin superfamily. ICAM-1 plays an important role in cell-cell interactions and contains five immunoglobulin-like domains. The minor group HRVs bind to members of the low-density lipoprotein receptor (LDL-R) family [5,6], which internalize LDL particles but also mediate the transport of macromolecules into cells by receptor-mediated endocytosis (Fig. 3). The ligand-binding amino terminus of the LDL receptors all contain various numbers of imperfect repeats of approximately 40 amino acids. These rigid ligand-binding domains are linked by four to five amino acids which confer some flexibility. Both ICAM-1 and the LDL receptors appear to bind their ligands by electrostatic interactions. HRV87 alone uses an unidentified sialoprotein as receptor [7] for which the receptor site is unknown. The major group HRV89 has the capacity to evolve under the pressure of passage in vitro to use an alternative receptor and even to infect cells devoid of its normal ICAM-1 receptor [8].

There is a remarkable difference in the location and accessibility of the receptor sites of the two groups of HRV. The major group HRV receptor site lies at the base of a depression or canyon around each five-fold axis [9] (Figs. 1 and 4). In contrast, the minor group HRV receptor binds to the star-shaped dome on the five-fold axis [10] (Figs. 1 and 4). The canyon hypothesis [1] proposed that the major group HRVs protect their receptor sites from immune surveillance by effectively hiding their receptor sites at the base of the canyon. The antibodies, being much larger than the ICAM-1 receptor, were supposed to be unable to reach the base of the narrow canyon. However, it was later shown that key viral amino acid residues involved in binding ICAM-1 are also accessible to antibodies [11]. Effectively, the receptor binding site is indeed accessible to antibodies but is flanked by residues capable of

Figure 1 Surface views of the reconstructed cryo-electron microscopy maps of (A) the minor group HRV2 and (B) the complex of HRV2 and a soluble fragment of the VLDL receptor where a "crown" of receptor molecules is seen on each five-fold axis. The icosahedral axes of one asymmetric unit are indicated in (A). Similar views show the major group HRV14 (C) and HRV16 (D) complexed with a soluble fragment of ICAM-1. All reconstructions are viewed down a two-fold axis. (Figures 1A and B are adapted from Hewat, E. A. et al., EMBO J., 19, 6317-6325, 2000; Figs. 1C and D are reproduced from Kolatkar, P. R. et al., EMBO J. 18, 6249-6259, 1999. With permission.)

Figure 1 Surface views of the reconstructed cryo-electron microscopy maps of (A) the minor group HRV2 and (B) the complex of HRV2 and a soluble fragment of the VLDL receptor where a "crown" of receptor molecules is seen on each five-fold axis. The icosahedral axes of one asymmetric unit are indicated in (A). Similar views show the major group HRV14 (C) and HRV16 (D) complexed with a soluble fragment of ICAM-1. All reconstructions are viewed down a two-fold axis. (Figures 1A and B are adapted from Hewat, E. A. et al., EMBO J., 19, 6317-6325, 2000; Figs. 1C and D are reproduced from Kolatkar, P. R. et al., EMBO J. 18, 6249-6259, 1999. With permission.)

mutating to give a viable virus that escapes immune surveillance [12]. This is an interesting example of a highly plausible hypothesis that is not quite correct.

Binding of ICAM-1 to major group HRVs, such asHRV14, initiates rapid uncoating at physiologic temperature without the need of any cellular machinery [13]. In contrast, binding of LDL receptors to minor group HRVs, such as HRV2, does not directly catalyze decapsidation [5], and the subsequent internalization into acidic endosomal compartments is required for the transfer of the viral RNA into the cytosol [14] (Fig. 3).

The difference in the stability of the virus-receptor complexes and in the receptor binding sites of the major and minor group HRVs correlate with differences in their uncoating mechanisms. Rossmann and colleagues [9] have

Figure 2 Schematic representation of the two proteins known to act as rhinovirus receptors. ICAM-1 contains five immunoglobulin-like domains and attaches to the major group HRVs by the N-terminal domain depicted in black. The ligand-binding amino terminus of the VLDL receptor (one of the LDL receptor family) contains eight imperfect repeats; two of these repeats bind to the minor group HRV2.

Figure 2 Schematic representation of the two proteins known to act as rhinovirus receptors. ICAM-1 contains five immunoglobulin-like domains and attaches to the major group HRVs by the N-terminal domain depicted in black. The ligand-binding amino terminus of the VLDL receptor (one of the LDL receptor family) contains eight imperfect repeats; two of these repeats bind to the minor group HRV2.

Figure 3 Schemas for attachment of soluble receptors to major and minor group HRVs and for the infection of cells by HRVs.

Figure 4 Schematic representation of the two step binding mechanism between ICAM-1 and the major group HRVs proposed by Kolatkar et al. [9]. See the text for a description of the first step shown in (A) and the second step in (B). Only two of the five ICAM-1 domains are shown. Part (C) shows how the VLDL receptor attaches to the minor group HRV2. Only three of the VLDL-R domains are shown. (Figures 4A and B are reproduced from Kolatkar, P. R. et al., EMBO J., 18, 6249-6259, 1999; Fig. 4C is adapted from Hewat, E. A. et al., EMBO J., 19, 6317-6325, 2000. With permission.)

Figure 4 Schematic representation of the two step binding mechanism between ICAM-1 and the major group HRVs proposed by Kolatkar et al. [9]. See the text for a description of the first step shown in (A) and the second step in (B). Only two of the five ICAM-1 domains are shown. Part (C) shows how the VLDL receptor attaches to the minor group HRV2. Only three of the VLDL-R domains are shown. (Figures 4A and B are reproduced from Kolatkar, P. R. et al., EMBO J., 18, 6249-6259, 1999; Fig. 4C is adapted from Hewat, E. A. et al., EMBO J., 19, 6317-6325, 2000. With permission.)

proposed that ICAM-1 binds to the major group HRVs in a two-step process, as shown in Fig. 4. In the first step, ICAM-1 binds essentially to the base and one side of the canyon in the conformation as observed in cryo-electron microscopy reconstructions. The second step would then consist of expulsion of the natural pocket factor, as the ICAM-1 molecule binds to the other side of the canyon. This would induce the VP1 to flex at the canyon, moving away from the five-fold axis and thus opening the pentameric vertex. Because the binding site of the HRV2 receptor lies entirely on the dome on the five-fold axis and does not overlap the canyon or the pocket in the canyon at all, the mechanism must be quite different. The binding of VLDL-R to HRV2 as seen by cryo-electron microscopy is probably also the first step in a two step process [10]. The first step of receptor binding simply ensures that the HRVs are anchored to the membrane. The second step (i.e., expulsion of the pocket factor and flexing of VP1 to open a passage for the exit of the molecule of RNA) is then triggered by the low pH (5.6) in the endosome (Fig. 4). It is generally believed that the RNA exits along one of the five-fold axes. As the capsid opens, the VP4 and the N-terminus of VP1 are externalized. It has been hypothesized that both VP4 and the N-terminus of VP1 are inserted into the membrane in order to facilitate passage of the RNA across the membrane [1].

Antiviral compounds, such as the "WIN compounds" produced by the former Sterling Winthrop Research Institute, bind in the VP1 pocket. In many major group viruses, this induces a deformation of the canyon which causes a loss of receptor binding. It also stabilizes the capsid; however, in minor group viruses these antivirals do not affect receptor binding [15], and their antiviral effect is based on their stabilizing effect only. This behavior is in accord with the fact that the binding site of LDL-R on minor group viruses does not overlap the pocket at the base of the canyon.

References

1. Rueckert, R. R. (1996). Picornaviridae: the viruses and their replication, in Fields, B. N., Knipe, D. M., and Howley, P. M., Eds., Fields Virology, pp. 609-654. Lippincott, Philadelphia, PA.

2. Rossmann, M. G., Arnold, E., Erickson, J. W., Frankenberger, E. A., Griffith, J. P., Hecht, H. J., Johnson, J. E., Kamer, G., Luo, M., Mosser, A. G., Rueckert, R. R., Sherry, B., and Vriend, G. (1985). Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317, 145-153.

3. Hadfield, A. T., Lee, W. M., Zhao, R., Oliveira, M. A., Minor, I., Rueckert, R. R., and Rossmann, M. G. (1997). The refined structure of human rhinovirus 16 at 2.15 angstrom resolution: implications for the viral life cycle. Structure 5, 427-441.

4. Greve, J. M., Davis, G., Meyer, A. M., Forte, C. P., Yost, S. C., Marlor, C. W., Kamarck, M. E., and McClelland, A. (1989). The major human rhinovirus receptor is ICAM-1. Cell 56, 839-847.

5. Gruenberger, M., Wandl, R., Nimpf, J., Hiesberger, T., Schneider, W. J., Kuechler, E., and Blaas, D. (1995). Avian homologs of the mammalian low-density lipoprotein receptor family bind minor receptor group human rhinovirus. J. Virol. 69, 7244-7247.

6. Hofer, F., Gruenberger, M., Kowalski, H., Machat, H., Huettinger, M., Kuechler, E., and Blaas, D. (1994). Members of the low density lipopro-tein receptor family mediate cell entry of a minor-group common cold virus. Proc. Natl. Acad. Sci. USA 91, 1839-1842.

7. Uncapher, C. R., DeWitt, C. M., and Colonno, R. J. (1991). The major and minor group receptor families contain all but one human rhinovirus serotype. Virology 180, 814-817.

8. Reischl, A., Reithmayer, M., Winsauer, G., Moser, R., Gosler, I., Blaas, D. (2001). Viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in ICAM-1-negative cells. J. Virol. 75, 9312-9319.

9. Kolatkar, P. R., Bella, J., Olson, N. H., Bator, C. M., Baker, T. S., and Rossmann, M. G. (1999). Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor. EMBO J. 18, 6249-6259.

10. Hewat, E. A., Neumann, E., Conway, J. F., Moser, R., Ronacher, B., Marlovits, T. C., and Blaas, D. (2000). The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view. EMBO J. 19, 6317-6325.

11. Smith, T. J., Chase, E. S., Schmidt, T. J., Olson, N. H., and Baker, T. S. (1996). Neutralizing antibody to human rhinovirus 14 penetrates the receptor-binding canyon. Nature 383, 350-354.

12. Hogle, J. M. (1993). The viral canyon. Curr. Biol. 3, 278-281.

13. Greve, J. M., Forte, C. P., Marlor, C. W., Meyer, A. M., Hooverlitty, H., Wunderlich, D., and McClelland, A. (1991). Mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of ICAM-1. J. Virol. 65, 6015-6023.

14. Neubauer, C., Frasel, L., Kuechler, E., and Blaas, D. (1987). Mechanism of entry of human rhinovirus 2 into HeLa cells. Virology 158, 255-258.

15. Kim, K. H., Willingmann, P., Gong, Z. X., Kremer, M. J., Chapman, M. S., Minor, I., Oliveira, M. A., Rossmann, M. G., Andries, K., Diana, G. D., Dutko, F. J., McKinley, M. A., and Pevear, D. C. (1993). A comparison of the anti-rhinoviral drug binding pocket in HRV14 and HRV1A. J. Mol. Biol. 230, 206-227.

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