Picornaviral Research Unit, Discipline of Immunology and Microbiology and 2Cancer Research Unit, Faculty of Medicine and Health Sciences, The University of Newcastle, Level 3, David Maddison Clinical Sciences Building, Royal Newcastle Hospital, Newcastle, 2300 New South Wales, Australia
Author for correspondence: Darren Shafren. Fax +61 2 4923 6814. e-mail dshafren{at}mail.newcastle.edu.au
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Abstract |
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CAV-21 lytic infection of cells expressing both ICAM-1 and DAF can only be completely inhibited by the combined action of anti-ICAM-1 (d1) and anti-DAF SCR1 MAb blockade (Shafren et al., 1997b ). Although, human rhinovirus 14 (HRV-14) and CAV-21 compete for the same binding epitope on ICAM-1 (Lonberg-Holm et al., 1976
), replication of HRV-14 in HeLa cells, in contrast to that of CAV-21, is completely inhibited by anti-ICAM-1 (d1) MAb blockade but is not affected by anti-DAF MAb pretreatment (Shafren et al., 1997b
). Interestingly, anti-DAF SCR3 MAb blockade partially blocks CAV-21 infection of HeLa cells without interfering with CAV-21 attachment to either DAF or ICAM-1 (Shafren et al., 1997b
). Taken together, these findings raise the interesting possibility that CAV-21 cell entry, unlike that of HRV-14, may be influenced by either extra- and/or intracellular interplay between DAF and ICAM-1.
In this study, using chimeric ICAM-1 receptors, we have shown that CAV-21 lytic cell infection mediated via the DAF/ICAM-1 receptor complex is not dependent on the presence of the transmembrane and cytoplasmic domains of ICAM-1. Secondly, employing chemical cross-linking, antibody and picornavirus binding techniques, we have confirmed that a close extracellular spatial association exists between DAF and ICAM-1 on the surface of ICAM-1-expressing RD cells and that this interaction is most likely to occur in an area on or proximal to DAF SCR3.
Firstly, we investigated the potential role of intracellular DAF/ICAM-1 interactions in the CAV-21 cell infection cycle. To address this question, a chimeric ICAM-1/CD36 molecule was constructed in which the fifth extracellular domain, the transmembrane and cytoplasmic domains of ICAM-1 were removed and replaced with the transmembrane and cytoplasmic domains from CD36. CD36 is an 88 kDa polypeptide and a cellular receptor for thrombospondin (Greenwalt et al., 1992 ). A schematic representation of wild-type ICAM-1, CD36 and chimeric ICAM-1/CD36 is shown in Fig. 1(a)
. To confirm the fidelity of surface-expressed chimeric ICAM-1/CD36, transfected RD cells were surface-labelled with biotin, immunoprecipitated with anti-ICAM-1 (Boyd et al., 1988
) or anti-CD36 (Mazurov et al., 1992
) MAbs where appropriate and analysed by SDSPAGE; specific proteins were visualized using enhanced chemiluminescence (Shafren et al., 1997b
). The results shown in Fig. 1(b)
reveal that ICAM-1 is expressed in RD cells as a 90 kDa polypeptide, and that CD36 and chimeric ICAM-1/CD36 are expressed as 85 and 70 kDa proteins, respectively. The identity of a second minor polypeptide in the ICAM-1/CD36 immunoprecipitate which possesses a similar molecular mass to native ICAM-1 has been shown to be a chimeric ICAM-1/CD36 molecule with a different glycosylation pattern compared to the major species (data not shown). The capacity of the anti-ICAM-1 MAb (d1) to efficiently immunoprecipitate the chimeric ICAM-1/CD36 molecule further confirms that the conformation of the d1 was retained despite the deletion of the fifth extracellular and the transmembrane/cytoplasmic domains.
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The fluorescence histograms shown in Fig. 1(c) indicate a comparable level of surface expression of ICAM-1 and chimeric ICAM-1/CD36. CD36 was expressed only on the surface of cells transfected with CD36 cDNA (Fig. 1c
). RD cells expressing ICAM-1 or chimeric ICAM-1/CD36 were highly susceptible to CAV-21-mediated cell lysis, whereas cells transfected with either vector alone (BOS) or CD36 were refractile to infection (Fig. 1d
).
Next, we investigated whether DAF and ICAM-1 shared an extracellular relationship on the surface of ICAM-1-expressing RD cells. Previously, we have shown a spatial association between ubiquitously surface-expressed DAF and ICAM-1 on HeLa cells (Shafren et al., 1997b ) and to address this question we transiently transfected DAF-expressing RD cells with either ICAM-1 or CD36 cDNA (Shafren et al., 1997b
). Cell transfectants were iodinated, chemically cross-linked and immunoprecipitated with anti-DAF (Kinoshita et al., 1985
), anti-ICAM-1 (Boyd et al., 1988
) or anti-CD36 MAbs (Mazurov et al., 1992
). In the ICAM-1-transfected RD cells, in the absence of cross-linking, the anti-DAF MAb and the anti-ICAM-1 MAb immunoprecipitated polypeptides of approximately 75 kDa and 90 kDa, respectively, whereas the anti-CD36 MAb did not recognize any proteins (Fig. 2a
). Following chemical cross-linking of these cells, immunoprecipitation with the anti-DAF MAb identified the DAF band at 75 kDa as previously seen in non-cross-linked samples, but also revealed an additional polypeptide of 90 kDa, similar to that observed in both ICAM-1 MAb immunoprecipitates. Immunoprecipitation of cross-linked cells with the anti-ICAM-1 MAb identified the ICAM-1 band at 90 kDa and a slight band at 75 kDa, similar to that observed in both anti-DAF immunoprecipitates. The finding that little detectable DAF was observed in the cross-linked anti-ICAM-1 immunoprecipitate may reflect the fact that DAF was expressed at a lower level than ICAM-1 on the surface of transfected RD cells (Fig. 2a
). Thus, a large proportion of cell surface ICAM-1 may not be associated with DAF. The DAF and ICAM-1 spatial association was shown to be specific and not an artefact of cell transfection, as CD36 MAb and DAF MAb immunoprecipitates of CD36-transfected RD cell lysates (cross-linked and non-cross-linked) revealed only the presence of polypeptides specific to the appropriate MAbs (Fig. 2b
).
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In this study, we have shown that CAV-21 lytically infects RD cells expressing either wild-type ICAM-1 or a chimeric form, in which the transmembrane and cytoplasmic domains are replaced with those from an unrelated molecule, CD36 (Fig. 1). This result is consistent with earlier findings in which HRV-14 and group B coxsackieviruses were shown to infect cells expressing a chimeric form of ICAM-1 and CAR, respectively, where the transmembrane and cytoplasmic domains were replaced with a glycosyl phosphatidylinositol anchor (Staunton et al., 1992
; Wang & Bergelson, 1999
). Overall, the data herein suggest that the postulated interplay between DAF and ICAM-1 in regulating CAV-21 cell entry probably occurs in an extracellular fashion rather than in an intracellular environment.
A close spatial association between DAF and ICAM-1 on the surface of ICAM-1-transfected RD cells was confirmed (Fig. 2). Antibody and virus binding patterns to surface-expressed DAF in the presence or absence of co-expressed ICAM-1 suggest that the interactions between DAF and ICAM-1 occur at an epitope located in or proximal to DAF SCR3 (Fig. 3c
). Molecular modelling studies have predicted that DAF is expressed on the cell surface in an S-shaped form (Kuttner-Kondo et al., 1996
) and, based on this structure, a schematic representation of the putative DAF/ICAM-1 spatial relationship is shown in Fig. 3(b)
. Previously, we have shown that anti-DAF SCR3 pretreatment of HeLa B cells does not inhibit CAV-21 cell binding but partially blocks CAV-21 replication (Shafren et al., 1997b
). The findings from this study that the DAF/ICAM-1 spatial relationship occurs at a region located near SCR3 of DAF suggest that DAF SCR3 MAb pretreatment may inhibit CAV-21 infection by impeding extracellular DAF/ICAM-1 interactions at this location.
HRV-14 and CAV-21 compete for the same epitope on ICAM-1 d1 (Lonberg-Holm et al., 1976 ), but while MAb blockade of this domain affords total protection against HRV-14 infection it does not do so for CAV-21 (Fig. 3a
). This finding raises the interesting possibility that when the ICAM-1-mediated route of cell entry is blocked, CAV-21, unlike HRV-14, may employ an alternate route of cell entry, possibility mediated via cross-linked-DAF (Shafren, 1998
). Studies employing HeLa cells expressing dominant-negative dynamin mutants lacking normal GTPase activity have confirmed that HRV-14 enters cells via a clathrin-coated pit endocytotic mechanism (De Tulleo & Kirchhausen, 1998
; Grunert et al., 1997
). However, the structurally similar poliovirus, using a cellular receptor from the same family as ICAM-1, employs a different cell entry mechanism (De Tulleo & Kirchhausen, 1998
).
Recently, simian virus 40 (SV-40) has been shown to enter cells via endocytotic structures known as caveolae (Anderson et al., 1996 ). Caveolae are specialized cell surface invaginations with several known functions, including endocytosis of macromolecules using mechanisms independent of clathrin-coated pits (Rothberg et al., 1990
; Sargiacomo et al., 1993
). This SV-40 route of cell internalization is mediated by the viral cross-linking of MHC class I molecules (Anderson et al., 1996
). Interestingly, DAF cross-linked by specific anti-DAF MAbs is sequestered to caveolae (Mayor et al., 1994
; Rothberg et al., 1992
). In contrast to HRV-14, CAV-21 binds to DAF SCR1 (Shafren et al., 1997b
; unpublished data) and can lytically infect RD cells in the absence of ICAM-1 when cell surface DAF is cross-linked with MAbs directed against DAF SCR2/3 (Shafren, 1998
). Therefore, as the putative DAF/ICAM-1 extracellular interaction is likely to occur on or proximal to DAF SCR3, one may postulate that ICAM-1 may in fact cross-link DAF, allowing translocation of DAF-bound CAV-21 to caveolae for subsequent cell internalization; this area is currently under investigation. Recent functional studies indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells (Henley et al., 1998
). Therefore, it would be of interest to investigate whether expression of a dominant-negative dynamin mutant in ICAM-1-transfected RD cells would inhibit CAV-21 cell infection.
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Acknowledgments |
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References |
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Received 2 August 1999;
accepted 6 December 1999.