Department of Microbiology and Immunology, The University of Melbourne, Parkville 3052, Victoria, Australia1
Department of Gastroenterology and Clinical Nutrition, The Royal Childrens Hospital, Parkville 3052, Victoria, Australia2
Author for correspondence: Barbara Coulson. Fax +61 3 9347 1540. e-mail b.coulson{at}microbiology.unimelb.edu.au
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Abstract |
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Introduction |
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The outermost layer of the non-enveloped, icosahedral virion is composed of the 37 kDa glycoprotein VP7 and spikes of the 88 kDa protein VP4, as dimers (Prasad et al., 1990 ), which extend about 12 nm above the VP7 surface (Prasad et al., 1988
; Yeager et al., 1994
). Both VP4 and VP7 independently elicit neutralizing, protective antibodies (Hoshino et al., 1995
; Offit et al., 1986
). VP4 is an important determinant of host cell tropism (Kalica et al., 1983
; Ramig & Galle, 1990
), virulence (Hoshino et al., 1995
), receptor binding and cell penetration (Kirkwood et al., 1998
; Ludert et al., 1996
). Proteolytic cleavage of VP4 into two subunits, VP8* (28 kDa) and VP5* (60 kDa) (Espejo et al., 1981
), results in increased infectivity (Clark et al., 1981
; Estes et al., 1981
) and rapid internalization of virus. VP7 may have a minor role in host cell entry (Ludert et al., 1996
).
A minority of animal rotaviruses, including the simian strain SA11 and the rhesus rotavirus RRV, bind to cell surface sialic acid (Fukudome et al., 1989 ) via VP4 (Mackow et al., 1989
), but this binding does not appear to be essential for infectivity of these viruses, as sialic acid-independent mutants retain their infectivity (Mendez et al., 1993
). Human rotaviruses do not utilize sialic acid for cellular attachment (Ciarlet & Estes, 1999
; Fukudome et al., 1989
). Cell lines fully permissive to human and monkey rotaviruses are monkey kidney epithelial (MA104) and human colonic adenocarcinoma (Caco-2, HT-29) types. However, HepG2 cells have been shown to support growth of hepatotropic rotaviruses (Ramig & Galle, 1990
).
Recently, we have shown that integrins are involved in rotavirus cell attachment and entry (Coulson et al., 1997 ; Hewish et al., 2000
). Integrins are
heterodimeric, transmembrane glycoproteins that are important in cell adhesion and signalling. Most human and animal rotaviruses (87%), including SA11, contain the amino acid sequence DGE at positions 308310 of VP5*. The peptide DGE(A) has been reported to act as a ligand in type I collagen for the
2
1 integrin (Staatz et al., 1991
). The VP7 of 43·7% of rotaviruses, including SA11 and RRV, contains the sequence LDV at aa 237239. The related sequences LDI and IDI are present in all mammalian rotaviruses at aa 269271 (Coulson et al., 1997
). Also, SA11, RRV and some human rotaviruses contain the sequence IDA at aa 538540 of VP4 (Hewish et al., 2000
). In the first connecting segment of the independently spliced IIICS domain of fibronectin, LDV is the minimal essential sequence for a major site of adhesion of fibronectin to the
4
1 and
4
7 integrins on a range of cell types (Komoriya et al., 1991
), and IDA is an
4 integrin ligand sequence in the C-terminal HepII domain of fibronectin. In addition, at aa 253255 in VP7, all mammalian rotaviruses contain the sequence GPR, which is a ligand for the
X
2 integrin in the N-terminal domain of fibrinogen (Loike et al., 1991
). In our experiments, peptides RDGEE and GPRP and monoclonal antibodies (MAbs) directed to
2,
4,
X,
1 and
2 integrin subunits blocked rotavirus infection specifically in an additive and dose-dependent manner (Coulson, 1997
; Coulson et al., 1997
; Hewish et al., 2000
). The ligand sequence in VP7 for
X
2 integrin, GPRP, is likely to be functional, since the GPRP peptide blocked SA11 and human rotavirus RV-5 infection of MA104 and Caco-2 cells (Coulson et al., 1997
). The role of the LDV and LDV-like sequences in VP7 is less clear, as VP4 of some rotavirus strains also contains the
4 integrin ligand sequence (IDA, see above), and the LDV-containing peptide has not yet been tested for blocking of virus binding or infection in cells expressing detectable
4 integrin. MAbs directed to
1,
3,
5,
6,
L,
M and
4 and RGD-containing peptides did not block SA11 rotavirus infection (Coulson, 1997
; Coulson et al., 1997
).
Most recently, we have shown that 2
1 and
4
1 integrins can act as cellular receptors for SA11, by studying SA11 attachment to and replication in K562 cells expressing
2
1 (
2-K562),
3
1 (
3-K562) or
4
1 (
4-K562) integrins on their surface as a result of transfection with integrin subunit cDNA (Hewish et al., 2000
). Levels of virus binding and infection in
2-K562 and
4-K562 cells were increased specifically over levels in
3-K562 and K562 cells. Additionally, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of
2
1 integrin, which correlated with further increases in the level of SA11 virus growth. Virus growth in
4-K562 cells that had also been induced to express
2
1 integrin with phorbol ester was to a level approaching that in MA104 cells (Hewish et al., 2000
).
One explanation for the restrictions on rotavirus replication in vitro is that expression of virus receptors, including 2
1 and
4
1, varies between cell lines. In order to examine this question, we have determined the levels of expression of integrins that have been demonstrated to be capable of acting as rotavirus receptors (
2
1 and
4
1) or implicated in rotavirus cell entry (
X
2) on a range of cell lines of human and monkey origin and correlated these levels with virus titres produced after infection with monkey and human rotavirus strains.
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Methods |
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Viruses.
The origins of monkey rotaviruses now designated as serotype P5B[2], G3 (SA11 and RRV), and human rotaviruses designated as serotypes P1A[8], G1 (RV-4, Wa), P3A, G1 (K8), P1B[4], G2 (RV-5), and P2A[6], G4 (ST-3), have been described previously (Coulson, 1993 ; Coulson et al., 1985
). Following activation of infectivity with 10 µg/ml porcine trypsin (Sigma) for 20 min at 37 °C, viruses were propagated in MA104 cells in the absence of serum and the presence of 1 µg/ml porcine trypsin as described previously (Hewish et al., 2000
; Sato et al., 1981
). Viruscell lysates were clarified by low-speed centrifugation and then stored at -70 °C. Titres of infectious virus were determined by indirect immunofluorescent staining of infected cells in MA104 cell monolayers inoculated with serial dilutions of the stocks (Coulson et al., 1985
).
MAbs.
Mouse MAbs to human integrin subunits used in flow cytometry were as follows: AK7 and RMAC11, directed against the chain (CD49b) of
2
1 integrin (Gamble et al., 1993
; OConnell et al., 1991
), from M. Berndt (Baker Medical Research Institute, Melbourne, Victoria, Australia) and A. DApice (St. Vincents Hospital, Melbourne, Victoria, Australia), respectively, as purified protein; P4C2 and P4G9 [hybridoma cell supernatant fluids (SNF)], directed against the
chain (CD49d) of
4
1 integrin (Kamata et al., 1995
), and P4C10 (purified protein) and QE2.E5 (hybridoma SNF), directed to integrin
1 chain (CD29), donated by D. Leavesley (Dept of Renal Medicine, The Royal Adelaide Hospital, Adelaide, South Australia) and E. Wayner (Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA); KB90 and 99.1.1.1, directed to the
chain (CD11c) of
X
2 integrin, from DAKO and P. Cameron, G. Stent and S. Sonza (Macfarlane Burnet Centre for Medical Research, Victoria, Australia), respectively; and MHM23 and AZN-L27, directed to integrin
2 chain (CD18), from DAKO and the Sixth International Workshop on Human Leucocyte Differentiation Antigens (Coulson, 1997
), respectively. Control MAbs were MOPC 21 (purified; Cappel, ICN Pharmaceuticals), ST-3:1 and ST-3:3 (hybridoma SNF directed to ST-3 rotavirus) and RV-5:2 (hybridoma SNF directed to RV-5 rotavirus) (Coulson, 1993
). Control MAbs were matched with test MAbs for isotype, diluent type and protein concentration.
Flow cytometric analysis.
Cell surface expression of integrins was detected by indirect immunofluorescent staining of 35x105 cells. Confluent RD, HepG2, Caco-2, COS-7 and MA104 cell monolayers were washed twice with PBS and cells were detached by incubation at 37 °C for 5 min in PBS containing 0·1% (w/v) trypsin (Difco) and 0·02% (w/v) EDTA (PBStrypsinEDTA). As trypsin treatment can produce proteolysis of the 4 integrin subunit (Hemler et al., 1987
), cells were detached by incubation for 10 min in PBS containing 0·75 mM EDTA (PBSEDTA) in some experiments. Detached cells were resuspended in DMEM containing 1% (v/v) FCS for 30 min at 37 °C with occasional gentle agitation to allow restitution of surface proteins and then the medium was replaced with PBS containing 1% (v/v) FCS and 0·1% (w/v) NaN3 (PBSFCSAz). K562 cells were washed twice in PBSFCSAz. Cells of all types were incubated for 45 min on ice with optimal dilutions of MAbs to integrin subunits or isotype-matched control MAbs diluted in PBSFCSAz. Optimal MAb dilutions were determined by testing serial dilutions of each MAb on each cell line. For the two-step stain, cells were washed once in PBSFCSAz, reacted for 45 min on ice with FITC-conjugated sheep anti-mouse F(ab')2 fragments (Silenus) diluted 1:50 in PBSFCSAz and then washed as before. For the three-step stain, cells were washed twice in PBSFCSAz and then reacted as before with biotin-conjugated sheep anti-mouse F(ab')2 fragments (Silenus) diluted 1:50 in PBSFCSAz. After two washes, cells were reacted as before with 3 µl per tube of undiluted phycoerythrin-conjugated streptavidin (Becton Dickinson) and then washed twice. Cells were fixed with 1% (v/v) ultrapure formaldehyde (Polysciences) in PBS before analysis of cellular fluorescence on a FACSort flow cytometer (Becton Dickinson). Viable cell populations were selected by gating dot plots of forward and side scatter and fluorescence intensity histograms of the gated cell populations were constructed. A positive relative linear median fluorescence intensity (RLMFI; median fluorescence intensity with anti-integrin MAb/median fluorescence intensity with control MAb) was defined as
1·20 (Wasserman et al., 1994
). All anti-integrin MAbs were tested at a range of dilutions and the data obtained at the optimal MAb dilution were used for calculation of the RLMFI value. The optimal MAb dilution was the highest dilution giving the maximum RLMFI value. All MAbs showed dose-dependent binding to the cell lines tested.
The antibody-binding capacity of adherent cell lines was determined by using the Quantum Simply Cellular kit (Flow Cytometry Standards Corp., San Juan, USA) as suggested by the supplier. The kit contains a mixture of microbeads, consisting of a blank and four populations that express different calibrated binding capacities for mouse IgG MAbs. The beads were treated identically with the test cell lines to derive a calibration curve from which the antibody-binding capacity of each cell line was obtained.
Rotavirus growth curve determination.
Confluent RD, HepG2, Caco-2, COS-7 and MA104 cell monolayers in 24-well plates (Nunclon) and K562 cells in exponential phase were washed twice with PBS. K562 cells were suspended in 1 ml aliquots in DMEM at 5x105 cells/ml. Cells of all types were incubated with trypsin-activated virus at multiplicities of infection (m.o.i.) of 0·110 for 1 h at 37 °C in 5% CO2/95% air. The inoculum was replaced with DMEM containing 1 µg/ml porcine trypsin and incubation was continued as appropriate. Infection was terminated by freezing at -70 °C and virus was released from cells by two further freezethaw cycles. After trypsin activation, titres of harvested virus were determined by indirect immunofluorescent staining of infected cells in MA104 cell monolayers inoculated with serial dilutions of the samples. Virus titres were expressed as the number of fluorescing cell-forming units (f.c.f.u.) per ml (Coulson et al., 1985 ).
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Results |
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Discussion |
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In contrast to human rotaviruses, SA11 was able to replicate to a low level in K562 cells, which did not express any integrin implicated in rotavirus cell entry. This suggests that another receptor(s), in addition to integrins, is used by SA11 in K562 cells, but that 2
1 and possibly
X
2 are required for efficient SA11 replication in cell culture. As SA11, but not human rotaviruses, can use sialic acid for cell attachment (Fukudome et al., 1989
), this is suggested to be the most likely candidate receptor for SA11 on K562 cells.
Our results are in full agreement with previous reports of the cell surface expression of these integrins. Of the 1 integrins, K562 cells have been shown to express only
5
1 (Hemler et al., 1987
) and were also found to lack
2 integrins, including
X
2 (Uciechowski & Schmidt, 1989
). On RD cells, 32% of the
1 expressed was as
4
1, whereas only 3% was as
2
1 (Hemler et al., 1987
). On HepG2 cells, 53% of the
1 expressed was as
2
1 but no
4
1 was detected, so RD cells expressed much less
2
1 than did HepG2 cells (Hemler et al., 1987
). Caco-2 and MA104 cells have been shown to express
2
1 (Basson et al., 1992
; Coulson et al., 1997
) and
2 (Coulson et al., 1997
). The expression of
2 integrins on non-lymphoid cells is controversial but, in humans,
2 integrins have been detected on rectal epithelial cells (Hussain et al., 1995
) and
X
2 has been found on isolated enterocytes (Martin-Villa et al., 1997
). Our studies showed that
X
2 detection in flow cytometric studies can depend on the use of a protease-free cell dissociation buffer, as has been reported previously for
4
1 detection (Hemler et al., 1987
).
Replication of rotaviruses in K562 and RD cells has not been studied previously, although growth studies with some human and monkey rotavirus strains in MA104, HepG2 and Caco-2 cells have been reported (Estes et al., 1979 ; Kitamoto et al., 1991
; Ramig & Galle, 1990
). Our SA11 growth curves in MA104 cells are similar to those reported previously (Estes et al., 1979
; Kitamoto et al., 1991
; Ramig & Galle, 1990
). Growth curves of SA11 in CV-1 cells (Estes et al., 1979
) were similar to our results in COS-7 cells, which are a derivative of the CV-1 line (Gluzman, 1981
). Levels of rotavirus growth in HepG2, Caco-2 and MA104 cells were shown to be determined by the origin of gene 4, encoding VP4 (Kirkwood et al., 1998
; Ludert et al., 1996
; Ramig & Galle, 1990
). Growth curves for RV-5, RV-4, K8 and ST-3 have not been reported previously.
Although there was an overall correlation between cellular expression of 2
1 (and possibly
X
2) and rotavirus replication, quantitative differences were discernible in the replication of different rotavirus strains in given cell lines. In particular, RD cells showed unusual relative growth curves for RRV, SA11 and human rotaviruses. SA11 replicated to a 100-fold lower titre in RD cells than did RRV, whereas these two strains have been reported to replicate to similar titres in MA104, Caco-2 and HepG2 cells (Kitamoto et al., 1991
). The difference between the maximum titres of human rotaviruses and of RRV in RD cells (1000-fold) was greater than that observed in MA104 cells (100-fold; Kitamoto et al., 1991
). Particularly for the human strains, which do not appear to use sialic acid, it is likely that additional cellular receptors exist. One candidate receptor may be
-D-galactose, as infection of cells with Wa and SA11 is blocked by the Ricinus communis agglutinin I (Jolly et al., 1999
; Superti & Donelli, 1995
).
Only RD cells expressed 4
1, so this integrin cannot be a necessary prerequisite for rotavirus infection. As
4 integrin expression is restricted mainly to cells of the immune system, it is likely that rotavirus may interact via
4 with these cells and modulate their function. Rotavirus infection in children results in a specific circulating memory T cell response that is mainly CD4+ and
4
7+. In the murine model, memory B cells providing the secretory IgA response and protective humoral immunity also express
4
7 (Franco & Greenberg, 1999
). It will be interesting to examine whether rotavirus can bind to or infect these
4
7+ B and T cells via
4 integrin.
In Caco-2 cells, but not in RD or MA104 cells, the X integrin subunit was more readily detected than was the
2 subunit. This pattern of apparently greater expression of
X than
2 has been observed previously for mononuclear cells in the intestine, which showed greater expression of each of the
2 integrin partners (
L,
M and
X) than of
2 (Bernstein et al., 1996
). In a study in which
2 expression was detected by using the same MAb used in our study (MHM23), small bowel lamina propria T cells also showed greater expression of
L than
2 (Smart et al., 1991
). It has been suggested that this may be due to conformational changes in the integrin heterodimer that result in
2 epitope masking (Smart et al., 1991
) or that another
partner for
L,
M and
X may exist (Bernstein et al., 1996
).
The role of X
2 integrin in rotavirus cell entry is not yet determined. Although expression of this integrin is generally considered to be restricted to immune cells, we have detected
X
2 on rotavirus-permissive cell lines and others have found
X
2 on enterocytes, so it is possible that
X
2 plays a role in rotavirus entry into permissive cells in vivo and in vitro. As
X
2 and
4
1 on neutrophils are important in adhesion of these cells at sites of inflammation and
X
2 is important for monocyte/macrophage and dendritic cell function and for homing of intraepithelial cells to the small intestine (Shibahara et al., 2000
), rotavirus interaction with
X
2 and/or
4
1 may affect these immune responses. Transfection of cells with integrins should provide a useful model for studying the requirements of rotavirus usage of integrins and other molecules during cell attachment and entry. Studies on rotavirus binding to and infection of
X
2-transfected K562 cells are in progress.
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Acknowledgments |
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Footnotes |
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c Present address: Centre for Animal Biotechnology, The University of Melbourne, Parkville 3052, Victoria, Australia.
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References |
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Received 9 March 2000;
accepted 7 June 2000.