Growth of rotaviruses in continuous human and monkey cell lines that vary in their expression of integrins

Sarah L. Londrigan1, Marilyn J. Hewish1, Melanie J. Thomsonb,1, Georgina M. Sandersc,1, Huseyin Mustafa2 and Barbara S. Coulson1,2

Department of Microbiology and Immunology, The University of Melbourne, Parkville 3052, Victoria, Australia1
Department of Gastroenterology and Clinical Nutrition, The Royal Children’s Hospital, Parkville 3052, Victoria, Australia2

Author for correspondence: Barbara Coulson. Fax +61 3 9347 1540. e-mail b.coulson{at}microbiology.unimelb.edu.au


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Rotavirus replication occurs in vivo in intestinal epithelial cells. Cell lines fully permissive to rotavirus include kidney epithelial (MA104), colonic (Caco-2) and hepatic (HepG2) types. Previously, it has been shown that cellular integrins {alpha}2{beta}1, {alpha}4{beta}1 and {alpha}X{beta}2 are involved in rotavirus cell entry. As receptor usage is a major determinant of virus tropism, the levels of cell surface expression of these integrins have now been investigated by flow cytometry on cell lines of human (Caco-2, HepG2, RD, K562) and monkey (MA104, COS-7) origin in relation to cellular susceptibility to infection with monkey and human rotaviruses. Cells supporting any replication of human rotaviruses (RD, HepG2, Caco-2, COS-7 and MA104) expressed {alpha}2{beta}1 and (when tested) {alpha}X{beta}2, whereas the non-permissive K562 cells did not express {alpha}2{beta}1, {alpha}4{beta}1 or {alpha}X{beta}2. Only RD cells expressed {alpha}4{beta}1. Although SA11 grew to higher titres in RD, HepG2, Caco-2, COS-7 and MA104 cells, this virus still replicated at a low level in K562 cells. In all cell lines tested, SA11 replicated to higher titres than did human strains, consistent with the ability of SA11 to use sialic acids as alternative receptors. Levels of cell surface {alpha}2 integrin correlated with levels of rotavirus growth. The {alpha}2 integrin relative linear median fluorescence intensity on K562, RD, COS-7, MA104 and Caco-2 cells correlated linearly with the titre of SA11 produced in these cells at 20 h after infection at a multiplicity of 0·1, and the data best fitted a sigmoidal dose–response curve (r2=1·00, P=0·005). Thus, growth of rotaviruses in these cell lines correlates with their surface expression of {alpha}2{beta}1 integrin and is consistent with their expression of {alpha}X{beta}2 and {alpha}4{beta}1 integrins.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Rotaviruses are double-stranded RNA viruses in the family Reoviridae and are the major cause of infantile gastroenteritis in most mammalian species. Primary replication of rotaviruses takes place in the mature enterocytes of the intestine.

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 {alpha}{beta} 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 308–310 of VP5*. The peptide DGE(A) has been reported to act as a ligand in type I collagen for the {alpha}2{beta}1 integrin (Staatz et al., 1991 ). The VP7 of 43·7% of rotaviruses, including SA11 and RRV, contains the sequence LDV at aa 237–239. The related sequences LDI and IDI are present in all mammalian rotaviruses at aa 269–271 (Coulson et al., 1997 ). Also, SA11, RRV and some human rotaviruses contain the sequence IDA at aa 538–540 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 {alpha}4{beta}1 and {alpha}4{beta}7 integrins on a range of cell types (Komoriya et al., 1991 ), and IDA is an {alpha}4 integrin ligand sequence in the C-terminal HepII domain of fibronectin. In addition, at aa 253–255 in VP7, all mammalian rotaviruses contain the sequence GPR, which is a ligand for the {alpha}X{beta}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 {alpha}2, {alpha}4, {alpha}X, {beta}1 and {beta}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 {alpha}X{beta}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 {alpha}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 {alpha}4 integrin. MAbs directed to {alpha}1, {alpha}3, {alpha}5, {alpha}6, {alpha}L, {alpha}M and {beta}4 and RGD-containing peptides did not block SA11 rotavirus infection (Coulson, 1997 ; Coulson et al., 1997 ).

Most recently, we have shown that {alpha}2{beta}1 and {alpha}4{beta}1 integrins can act as cellular receptors for SA11, by studying SA11 attachment to and replication in K562 cells expressing {alpha}2{beta}1 ({alpha}2-K562), {alpha}3{beta}1 ({alpha}3-K562) or {alpha}4{beta}1 ({alpha}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 {alpha}2-K562 and {alpha}4-K562 cells were increased specifically over levels in {alpha}3-K562 and K562 cells. Additionally, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of {alpha}2{beta}1 integrin, which correlated with further increases in the level of SA11 virus growth. Virus growth in {alpha}4-K562 cells that had also been induced to express {alpha}2{beta}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 {alpha}2{beta}1 and {alpha}4{beta}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 ({alpha}2{beta}1 and {alpha}4{beta}1) or implicated in rotavirus cell entry ({alpha}X{beta}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.


   Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell lines.
Caco-2 cells were obtained from the ATCC. Human rhabdomyosarcoma (RD) and hepatoma (HepG2) cells were respectively provided by C. Birch and H. Siow of the Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria, Australia. COS-7 cells were derived originally from the African green monkey kidney epithelial cell line CV-1 (Gluzman, 1981 ) and were provided by M. Sandrin, The Austin Research Institute, Austin and Repatriation Medical Centre, Heidelberg, Victoria, Australia. African green monkey epithelial (MA104), COS-7, RD and HepG2 cells were grown in Dulbecco’s modification of Eagles’ medium including 2 mM L-glutamine (Gibco), 20 mM HEPES (Boehringer Mannheim), 26·6 µg/ml gentamicin (Cidomycin; Roussel) and 2 µg/ml Fungizone (Gibco) (DMEM), supplemented with 10% (v/v) heat-inactivated FCS (Commonwealth Serum Laboratories, Parkville, Victoria, Australia). Caco-2 cells were grown in modified DMEM (Trace Biosciences), which contained 0·075% (w/v) sodium bicarbonate and 20% (v/v) FCS. Human non-adherent myelogenous leukaemic cells (K562) were provided by I. Bertoncello, Peter McCallum Cancer Research Institute, Melbourne, Victoria, Australia, and grown in DMEM containing 20% (v/v) FCS.

{blacksquare} 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 ). Virus–cell 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 ).

{blacksquare} MAbs.
Mouse MAbs to human integrin subunits used in flow cytometry were as follows: AK7 and RMAC11, directed against the {alpha} chain (CD49b) of {alpha}2{beta}1 integrin (Gamble et al., 1993 ; O’Connell et al., 1991 ), from M. Berndt (Baker Medical Research Institute, Melbourne, Victoria, Australia) and A. D’Apice (St. Vincent’s Hospital, Melbourne, Victoria, Australia), respectively, as purified protein; P4C2 and P4G9 [hybridoma cell supernatant fluids (SNF)], directed against the {alpha} chain (CD49d) of {alpha}4{beta}1 integrin (Kamata et al., 1995 ), and P4C10 (purified protein) and QE2.E5 (hybridoma SNF), directed to integrin {beta}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 {alpha} chain (CD11c) of {alpha}X{beta}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 {beta}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.

{blacksquare} Flow cytometric analysis.
Cell surface expression of integrins was detected by indirect immunofluorescent staining of 3–5x105 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 (PBS–trypsin–EDTA). As trypsin treatment can produce proteolysis of the {alpha}4 integrin subunit (Hemler et al., 1987 ), cells were detached by incubation for 10 min in PBS containing 0·75 mM EDTA (PBS–EDTA) 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 (PBS–FCS–Az). K562 cells were washed twice in PBS–FCS–Az. 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 PBS–FCS–Az. 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 PBS–FCS–Az, reacted for 45 min on ice with FITC-conjugated sheep anti-mouse F(ab')2 fragments (Silenus) diluted 1:50 in PBS–FCS–Az and then washed as before. For the three-step stain, cells were washed twice in PBS–FCS–Az and then reacted as before with biotin-conjugated sheep anti-mouse F(ab')2 fragments (Silenus) diluted 1:50 in PBS–FCS–Az. 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.

{blacksquare} 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 0·1–10 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 freeze–thaw 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 ).


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Cell surface expression of integrins
Fig. 1 shows fluorescence intensity histograms determined by flow cytometry for the expression of integrin subunits {alpha}2, {alpha}4, {beta}1, {alpha}X and {beta}2 on the surface of human non-adherent K562 cells, on human adherent cell lines RD, HepG2 and Caco-2 and on monkey kidney cell lines MA104 and COS-7. All cell lines tested (K562, HepG2, Caco-2, COS-7 and MA104) expressed {beta}1 integrins. Integrin {alpha}2{beta}1 was expressed by all adherent cell lines tested (RD, HepG2, COS-7 and MA104) and we have shown previously that Caco-2 cells express {alpha}2{beta}1 (Coulson et al., 1997 ). Integrin subunits {alpha}X and {beta}2 were also expressed by all adherent cell lines tested. On RD cells, {alpha}X and {beta}2 were detectable by a two-step stain, whereas a three-step stain was required for detection of {alpha}X on Caco-2 and MA104 cells and for detection of {beta}2 on HepG2, Caco-2, COS-7 and MA104 cells. This suggests that the latter cell lines express lower levels of {alpha}X{beta}2 than do RD cells. Expression of {alpha}X was detected on MA104 cell suspensions produced by using PBS–EDTA but not on those produced with PBS–trypsin–EDTA (Fig. 1b), showing that the extracellular domain of the {alpha}X subunit is sensitive to digestion by trypsin. Only RD cells expressed {alpha}4{beta}1 integrin. K562 cells expressed none of the integrins implicated in rotavirus cell entry.




View larger version (81K):
[in this window]
[in a new window]
 
Fig. 1. Representative flow cytometric histograms of the surface expression of integrin subunits {alpha}2, {alpha}4 and {beta}1 (a) and {alpha}X and {beta}2 (b) on K562, RD, HepG2, Caco-2, MA104 and COS-7 cells. The cell line analysed in each panel is indicated. Test MAb histograms are shown in bold and control MAb traces are shown as faint lines. Control MAbs used varied, as each was matched with the test MAb for isotype and diluent. Adherent cell lines were detached with PBS–trypsin–EDTA unless otherwise indicated. Panels showing studies in which adherent cells were detached using PBS–EDTA rather than PBS–trypsin–EDTA are labelled ‘EDTA’. Expression of {alpha}2 was determined by using 10 µg/ml MAbs RMAC11 (anti-{alpha}2) and MOPC21 (control) in a three-step stain (for K562 cells), 10 µg/ml MAbs AK7 (anti-{alpha}2) and MOPC21 in a two-step stain (for HepG2 and COS-7 cells) or 20 µg/ml MAb RMAC11 and ST-3:3 (control) in a two-step stain (for MA104 cells). Expression of {alpha}4 was determined by using MAbs P4G9 (anti-{alpha}4) and RV-5:2 as control (1:2 dilution of hybridoma SNF) with a three-step stain. Expression of {beta}1 was determined with two-step stains using MAbs QE2.E5 (anti-{beta}1) and RV-5:2 for K562 cells and using 20 µg/ml MAbs P4C10 (anti-{beta}1) and MOPC21 for RD, HepG2 and MA104 cells. Expression of {beta}1 on Caco-2 cells was determined by using both MAb QE2.E5 (Caco-2A) and MAb P4C10 (Caco-2B). The RLMFI values obtained with these two {beta}1 MAbs were similar. Expression of {alpha}X was determined by using 80 µg/ml MAbs KB90 (anti-{alpha}X) and MOPC21 in a three-step stain (for K562 cells) or 40 µg/ml MAbs KB90 and MOPC21 in a two-step stain (for RD cells) or a three-step stain (for Caco-2 and MA104 cells). Expression of {beta}2 was determined by using 69 µg/ml MAbs MHM23 (anti-{beta}2) and MOPC21 in a two-step stain (for K562 and RD cells) or 35 µg/ml MAbs MHM23 and MOPC21 in a three-step stain (for the remaining cell lines).

 
The levels of integrin expression on K562 and RD cells are shown in Table 1. {alpha}4 and {alpha}X integrin subunits were not detected on K562 cells, even with the more sensitive three-step stain and two alternative MAbs. In a three-step stain, the {alpha}2 subunit was also not detected on K562 cells. In addition, the {beta}2 subunit was also not detected on these cells with two alternative MAbs in a two-step stain. In contrast, both the MAbs to {alpha}4 bound RD cells specifically.


View this table:
[in this window]
[in a new window]
 
Table 1. Surface expression of integrins on K562 and RD cells

 
Table 2 provides the levels of integrin expression on HepG2, Caco-2, COS-7 and MA104 cells. Human cell lines HepG2 and Caco-2 showed higher RLMFI values with the anti-{alpha}2 MAb AK7 than did monkey cell lines COS-7 and MA104. This pattern was also evident in the antibody-binding capacities of these cells with this MAb. All of these cell lines showed at least 13600 antibody-binding sites per cell for MAb AK7, but HepG2 and Caco-2 cells showed more sites than did COS-7 and MA104 cells. Although it is possible that MAb AK7, being directed to human {alpha}2, does not bind as well to monkey as to human {alpha}2, no evidence for this has been published to our knowledge; thus, comparisons between levels of integrin expression on human and monkey cell lines using this MAb are likely to be reliable.


View this table:
[in this window]
[in a new window]
 
Table 2. Surface expression of integrins on HepG2, Caco-2, COS-7 and MA104 cells

 
Growth of rotaviruses in K562, RD, COS-7 and MA104 cells
Growth curves of simian rotavirus SA11 and human rotavirus RV-5 in K562 and MA104 cells at a range of m.o.i. are shown in Fig. 2. Irrespective of the m.o.i., the maximum titres of SA11 produced in K562 cells were only 14- to 31-fold higher than the input virus titre (titre of virus associated with cells after incubation of virus with cells for 1 h at 37 °C). In contrast, the maximum titre of SA11 produced in MA104 cells at the low m.o.i. of 0·1 was 925000-fold higher than the input virus titre. The maximum RV-5 rotavirus titres recorded in K562 cells were only 0·67- to 0·99-fold of the input virus titre, whereas the maximum titre produced in MA104 cells was 440- to 1520-fold higher than the input virus titre. Thus, SA11 showed very limited replication in K562 cells and RV-5 did not replicate at all. In MA104 cells, SA11 at an m.o.i. of 0·1 grew to a 2000-fold higher titre than did RV-5 at an m.o.i. of 0·2.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. Rotavirus growth in K562 and MA104 cells at a range of m.o.i. SA11 infection of MA104 cells (m.o.i.=0·1) and K562 cells (m.o.i.=0·1, 1, 10) and RV-5 infection of MA104 and K562 cells (m.o.i.=0·2, 1, 5) are shown. Bars represent 95% confidence intervals (CI) of the mean. p.i., Post-infection.

 
Growth curves of SA11, RRV and human rotaviruses RV-4 and K8 in RD cells are shown in Fig. 3. Depending on the m.o.i. used, the maximum titres of SA11 produced were 132- to 759-fold higher than the input virus titre and those of RRV were 788- to 14600-fold higher than the input titres. Thus, RD cells are permissive to SA11 and RRV. In contrast, RV-4 titres increased only 1·0- to 1·7-fold over input virus titre and K8 grew to titres 8·1- to 38-fold higher than input virus titres. RD cells are resistant to RV-4 infection but allowed low levels of K8 rotavirus replication.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. Rotavirus growth in RD and MA104 cells at a range of m.o.i. SA11 infection of MA104 cells (m.o.i.=0·1) and of RD cells (m.o.i.=0·1, 10), RRV and RV-4 infection of RD cells (m.o.i.=0·1, 1) and K8 infection of RD cells (m.o.i.=0·1, 0·5, 1) are shown. Bars represent 95% CI.

 
Growth curves of SA11, RV-5, Wa and ST-3 rotaviruses in COS-7 cells are shown in Fig. 4. All rotavirus strains tested replicated in these cells, with maximum titres being 800-fold above input virus titre for SA11, 92-fold above input for RV-5, 15-fold above input for Wa and 2·3-fold above input for ST-3. Thus, in COS-7 cells at an m.o.i. of 1·0, RV-5 grew to a higher titre than did Wa and ST-3 replicated to a low titre only. In MA104 cells, Wa rotavirus grew to a maximum titre 1760-fold above the input virus titre and ST-3 grew to a maximum titre 34-fold above the input titre. All rotavirus strains tested grew to significantly lower titre in COS-7 cells than in MA104 cells.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4. Growth of rotavirus strains SA11, RV-5, Wa and ST-3 in COS-7 and MA104 cells. M.o.i.=1. Bars represent 95% CI.

 
Comparison of cellular expression of integrins with cellular susceptibility to infection with monkey and human rotaviruses
As shown in Table 3, the cell line that did not express any integrin implicated in rotavirus cell entry (K562) was not susceptible to RV-5 rotavirus infection and showed the lowest titre of SA11 at 20 h post-infection (p.i.) (m.o.i.=0·1) of any cell line tested. On RD cells, expression of {alpha}2 integrin is just detectable by flow cytometry (Chan et al., 1991 ; Hemler et al., 1987 ), giving an RLMFI of 1·3, and we showed that these cells do express {alpha}4{beta}1 and {alpha}X{beta}2. SA11 replicated to a 1·2-fold higher titre in RD cells than in K562 cells and human strains replicated to a low level. Higher levels of {alpha}2 integrin were expressed on Caco-2, COS-7 and MA104 cells, all of which supported both SA11 and human rotavirus infection at moderate to high levels. Expression of no, low or high levels of {alpha}2 integrin correlated with no, low and moderate levels of human rotavirus replication. The absence of {alpha}X{beta}2 also correlated with the inability of RV-5 to grow in K562 cells and with the low level of SA11 replication in these cells. Overall, all cells expressing {alpha}X{beta}2 were susceptible to rotavirus infection to varying degrees.


View this table:
[in this window]
[in a new window]
 
Table 3. Comparison of cellular integrin expression with susceptibility to rotavirus infection

 
The correlation between the {alpha}2 integrin RLMFI value and the titre of SA11 produced at 20 h p.i. after infection at an m.o.i. of 0·1 was analysed for K562, RD, COS-7, MA104 and Caco-2 cells (Fig. 5). There was a highly significant correlation between these parameters by the two-tailed Pearson test (P=0·005) and the data were best fitted by a sigmoidal dose-response curve with variable slope (r2=1·00). This suggests that SA11 may be binding allosterically to {alpha}2 integrin as a result of the interaction of multimeric virus-binding sites with increasing numbers of receptors. The titres of SA11 produced after one cycle of virus replication correlated quantitatively with the level of cellular expression of {alpha}2 integrin, and cellular levels of {alpha}2 integrin expression may therefore be important in determining SA11 rotavirus yields.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Correlation of growth of SA11 rotavirus with cellular {alpha}2 integrin expression. The data for SA11 growth (expressed as the mean titre±SD of virus produced at 20 h p.i. from cells infected at an m.o.i. of 0·1) and {alpha}2 integrin expression (expressed as the log10 of the RLMFI) taken from Tables 1–3 for K562, RD, COS-7, MA104 and Caco-2 cells are represented as open squares. The line shows the best fit determined by non-linear regression analysis (r2=1·00) and corresponds to a sigmoidal dose-response curve with variable slope.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
If integrins play an important role in rotavirus entry into cells, their cellular expression should be necessary for the cells to be fully permissive to rotavirus infection. In addition, levels of cellular integrin expression might be expected to correlate with levels of rotavirus replication. In this study, we have shown that cell lines that allow any replication of human rotaviruses express {alpha}2{beta}1 integrin and (when tested) {alpha}X{beta}2 integrin, whereas the K562 cell line, which was totally non-permissive to human rotavirus RV-5 infection, did not express these integrins or the other integrin implicated as a rotavirus receptor, {alpha}4{beta}1. The level of human rotavirus replication, within broad ranges of virus replication index (VRI), correlated positively with the level of expression of {alpha}2 integrin subunit on human cell lines. Thus, cellular expression of {alpha}2{beta}1, and probably {alpha}X{beta}2, is consistent with these integrins being important for human rotavirus cell entry. Titres of simian rotavirus SA11 produced 20 h after infection at an m.o.i. of 0·1 correlated quantitatively with levels of expression of the {alpha}2 integrin, expressed as an RLMFI. This correlation of {alpha}2{beta}1 expression with SA11 yield provides further support for the role for this integrin as a rotavirus receptor. A similar correlation between SA11 rotavirus yields and the {alpha}2 integrin RLMFI was observed for K562 cells transfected with {alpha}2 integrin and/or expressing additional {alpha}2 integrin due to phorbol ester treatment (Hewish et al., 2000 ).

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 {alpha}2{beta}1 and possibly {alpha}X{beta}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 {beta}1 integrins, K562 cells have been shown to express only {alpha}5{beta}1 (Hemler et al., 1987 ) and were also found to lack {beta}2 integrins, including {alpha}X{beta}2 (Uciechowski & Schmidt, 1989 ). On RD cells, 32% of the {beta}1 expressed was as {alpha}4{beta}1, whereas only 3% was as {alpha}2{beta}1 (Hemler et al., 1987 ). On HepG2 cells, 53% of the {beta}1 expressed was as {alpha}2{beta}1 but no {alpha}4{beta}1 was detected, so RD cells expressed much less {alpha}2{beta}1 than did HepG2 cells (Hemler et al., 1987 ). Caco-2 and MA104 cells have been shown to express {alpha}2{beta}1 (Basson et al., 1992 ; Coulson et al., 1997 ) and {beta}2 (Coulson et al., 1997 ). The expression of {beta}2 integrins on non-lymphoid cells is controversial but, in humans, {beta}2 integrins have been detected on rectal epithelial cells (Hussain et al., 1995 ) and {alpha}X{beta}2 has been found on isolated enterocytes (Martin-Villa et al., 1997 ). Our studies showed that {alpha}X{beta}2 detection in flow cytometric studies can depend on the use of a protease-free cell dissociation buffer, as has been reported previously for {alpha}4{beta}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 {alpha}2{beta}1 (and possibly {alpha}X{beta}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 {beta}-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 {alpha}4{beta}1, so this integrin cannot be a necessary prerequisite for rotavirus infection. As {alpha}4 integrin expression is restricted mainly to cells of the immune system, it is likely that rotavirus may interact via {alpha}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 {alpha}4{beta}7+. In the murine model, memory B cells providing the secretory IgA response and protective humoral immunity also express {alpha}4{beta}7 (Franco & Greenberg, 1999 ). It will be interesting to examine whether rotavirus can bind to or infect these {alpha}4{beta}7+ B and T cells via {alpha}4 integrin.

In Caco-2 cells, but not in RD or MA104 cells, the {alpha}X integrin subunit was more readily detected than was the {beta}2 subunit. This pattern of apparently greater expression of {alpha}X than {beta}2 has been observed previously for mononuclear cells in the intestine, which showed greater expression of each of the {beta}2 integrin partners ({alpha}L, {alpha}M and {alpha}X) than of {beta}2 (Bernstein et al., 1996 ). In a study in which {beta}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 {alpha}L than {beta}2 (Smart et al., 1991 ). It has been suggested that this may be due to conformational changes in the integrin heterodimer that result in {beta}2 epitope masking (Smart et al., 1991 ) or that another {beta} partner for {alpha}L, {alpha}M and {alpha}X may exist (Bernstein et al., 1996 ).

The role of {alpha}X{beta}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 {alpha}X{beta}2 on rotavirus-permissive cell lines and others have found {alpha}X{beta}2 on enterocytes, so it is possible that {alpha}X{beta}2 plays a role in rotavirus entry into permissive cells in vivo and in vitro. As {alpha}X{beta}2 and {alpha}4{beta}1 on neutrophils are important in adhesion of these cells at sites of inflammation and {alpha}X{beta}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 {alpha}X{beta}2 and/or {alpha}4{beta}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 {alpha}X{beta}2-transfected K562 cells are in progress.


   Acknowledgments
 
We are very grateful to M. Sandrin, C. Birch, I. Bertoncello and H. Siow for provision of cell lines, to M. Berndt, A. D’Apice, D. Leavesley, E. Wayner, P. Cameron, G. Stent, Y. van Kooyk and S. Sonza for gifts of monoclonal antibodies and to F. Battye for advice on determining cellular antibody binding capacity. This work was supported by project grants 940315 and 980635 from the National Health and Medical Research Council of Australia.


   Footnotes
 
b Present address: Department of Immunobiology, Institute of Child Health, Great Ormond St. Hospital, London, UK.

c Present address: Centre for Animal Biotechnology, The University of Melbourne, Parkville 3052, Victoria, Australia.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Basson, M. D., Modlin, I. M. & Madri, J. A. (1992). Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. Journal of Clinical Investigation 90, 15-23.[Medline]

Bernstein, C. N., Sargent, M., Gallatin, W. M. & Wilkins, J. (1996). Beta 2-integrin/intercellular adhesion molecule (ICAM) expression in the normal human intestine. Clinical and Experimental Immunology 106, 160-169.[Medline]

Chan, B. M., Matsuura, N., Takada, Y., Zetter, B. R. & Hemler, M. E. (1991). In vitro and in vivo consequences of VLA-2 expression on rhabdomyosarcoma cells. Science 251, 1600-1602.[Medline]

Ciarlet, M. & Estes, M. K. (1999). Human and most animal rotavirus strains do not require the presence of sialic acid on the cell surface for efficient infectivity. Journal of General Virology 80, 943-948.[Abstract]

Clark, S. M., Roth, J. R., Clark, M. L., Barnett, B. B. & Spendlove, R. S. (1981). Trypsin enhancement of rotavirus infectivity: mechanism of enhancement. Journal of Virology 39, 816-822.[Medline]

Coulson, B. S. (1993). Typing of human rotavirus VP4 by an enzyme immunoassay using monoclonal antibodies. Journal of Clinical Microbiology 31, 1-8.[Abstract]

Coulson, B. S. (1997). Effects of Workshop monoclonal antibodies on rotavirus infection of cells. In Leucocyte Typing VI, pp. 391-393. Edited by T. Kishimoto, H. Kikutani, A. E. G. Kr. von dem Borne, S. M. Goyert, D. Y. Mason, M. Miyasaka, L. Moretta, K. Okumura, S. Shaw, T. A. Springer, K. Sugamura & H. Zola. New York: Garland Publishing.

Coulson, B. S., Fowler, K. J., Bishop, R. F. & Cotton, R. G. (1985). Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates. Journal of Virology 54, 14-20.[Medline]

Coulson, B. S., Londrigan, S. L. & Lee, D. J. (1997). Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells. Proceedings of the National Academy of Sciences, USA 94, 5389-5394.[Abstract/Free Full Text]

Espejo, R. T., Lopez, S. & Arias, C. (1981). Structural polypeptides of simian rotavirus SA11 and the effect of trypsin. Journal of Virology 37, 156-160.[Medline]

Estes, M. K., Graham, D. Y., Gerba, C. P. & Smith, E. M. (1979). Simian rotavirus SA11 replication in cell cultures. Journal of Virology 31, 810-815.[Medline]

Estes, M. K., Graham, D. Y. & Mason, B. B. (1981). Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. Journal of Virology 39, 879-888.[Medline]

Franco, M. A. & Greenberg, H. B. (1999). Immunity to rotavirus infection in mice. Journal of Infectious Diseases 179 (Suppl. 3), S466–S469.

Fukudome, K., Yoshie, O. & Konno, T. (1989). Comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption. Virology 172, 196-205.[Medline]

Gamble, J. R., Matthias, L. J., Meyer, G., Kaur, P., Russ, G., Faull, R., Berndt, M. C. & Vadas, M. A. (1993). Regulation of in vitro capillary tube formation by anti-integrin antibodies. Journal of Cell Biology 121, 931-943.[Abstract]

Gluzman, Y. (1981). SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175-182.[Medline]

Hemler, M. E., Huang, C., Takada, Y., Schwarz, L., Strominger, J. L. & Clabby, M. L. (1987). Characterization of the cell surface heterodimer VLA-4 and related peptides. Journal of Biological Chemistry 262, 11478-11485.[Abstract/Free Full Text]

Hewish, M. J., Takada, Y. & Coulson, B. S. (2000). Integrins {alpha}2{beta}1 and {alpha}4{beta}1 can mediate SA11 rotavirus attachment and entry into cells. Journal of Virology 74, 228-236.[Abstract/Free Full Text]

Hoshino, Y., Saif, L. J., Kang, S. Y., Sereno, M. M., Chen, W. K. & Kapikian, A. Z. (1995). Identification of group A rotavirus genes associated with virulence of a porcine rotavirus and host range restriction of a human rotavirus in the gnotobiotic piglet model. Virology 209, 274-280.[Medline]

Hussain, L. A., Kelly, C. G., Rodin, A., Jourdan, M. & Lehner, T. (1995). Investigation of the complement receptor 3 (CD11b/CD18) in human rectal epithelium. Clinical and Experimental Immunology 102, 384-388.[Medline]

Jolly, C., Beisner, B. & Holmes, I. (1999). Rotavirus infection of MA104 cells is inhibited by Ricinus lectin and separately expressed single binding domains. In Abstracts of the XIth International Congress of Virology, VW 25.05, p. 149. Sydney, Australia.

Kalica, A. R., Flores, J. & Greenberg, H. B. (1983). Identification of the rotaviral gene that codes for hemagglutination and protease-enhanced plaque formation. Virology 125, 194-205.[Medline]

Kamata, T., Puzon, W. & Takada, Y. (1995). Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29). Biochemical Journal 305, 945-951.[Medline]

Kirkwood, C. D., Bishop, R. F. & Coulson, B. S. (1998). Attachment and growth of human rotaviruses RV-3 and S12/85 in Caco-2 cells depend on VP4. Journal of Virology 72, 9348-9352.[Abstract/Free Full Text]

Kitamoto, N., Ramig, R. F., Matson, D. O. & Estes, M. K. (1991). Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2). Virology 184, 729-737.[Medline]

Komoriya, A., Green, L. J., Mervic, M., Yamada, S. S., Yamada, K. M. & Humphries, M. J. (1991). The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine–aspartic acid–valine. Journal of Biological Chemistry 266, 15075-15079.[Abstract/Free Full Text]

Loike, J. D., Sodeik, B., Cao, L., Leucona, S., Weitz, J. I., Detmers, P. A., Wright, S. D. & Silverstein, S. C. (1991). CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen. Proceedings of the National Academy of Sciences, USA 88, 1044-1048.[Abstract]

Ludert, J. E., Feng, N., Yu, J. H., Broome, R. L., Hoshino, Y. & Greenberg, H. B. (1996). Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo. Journal of Virology 70, 487-493.[Abstract]

Mackow, E. R., Barnett, J. W., Chan, H. & Greenberg, H. B. (1989). The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant. Journal of Virology 63, 1661-1668.[Medline]

Martin-Villa, J. M., Ferre-Lopez, S., Lopez-Suarez, J. C., Corell, A., Perez-Blas, M. & Arnaiz-Villena, A. (1997). Cell surface phenotype and ultramicroscopic analysis of purified human enterocytes: a possible antigen-presenting cell in the intestine. Tissue Antigens 50, 586-592.[Medline]

Mendez, E., Arias, C. F. & Lopez, S. (1993). Binding to sialic acids is not an essential step for the entry of animal rotaviruses to epithelial cells in culture. Journal of Virology 67, 5253-5259.[Abstract]

O’Connell, P. J., Faull, R., Russ, G. R. & D’Apice, A. J. (1991). VLA-2 is a collagen receptor on endothelial cells. Immunology and Cell Biology 69, 103-110.[Medline]

Offit, P. A., Blavat, G., Greenberg, H. B. & Clark, H. F. (1986). Molecular basis of rotavirus virulence: role of gene segment 4. Journal of Virology 57, 46-49.[Medline]

Prasad, B. V., Wang, G. J., Clerx, J. P. & Chiu, W. (1988). Three-dimensional structure of rotavirus. Journal of Molecular Biology 199, 269-275.[Medline]

Prasad, B. V., Burns, J. W., Marietta, E., Estes, M. K. & Chiu, W. (1990). Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy. Nature 343, 476-479.[Medline]

Ramig, R. F. & Galle, K. L. (1990). Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (HepG2). Journal of Virology 64, 1044-1049.[Medline]

Sato, K., Inaba, Y., Shinozaki, T., Fujii, R. & Matumoto, M. (1981). Isolation of human rotavirus in cell cultures: brief report. Archives of Virology 69, 155-160.[Medline]

Shibahara, T., Si-Tahar, M., Shaw, S. K. & Madara, J. L. (2000). Adhesion molecules expressed on homing lymphocytes in model intestinal epithelia. Gastroenterology 118, 289-298.[Medline]

Smart, C. J., Calabrese, A., Oakes, D. J., Howdle, P. D. & Trejdosiewicz, L. K. (1991). Expression of the LFA-1 beta 2 integrin (CD11a/CD18) and ICAM-1 (CD54) in normal and coeliac small bowel mucosa. Scandinavian Journal of Immunology 34, 299-305.[Medline]

Staatz, W. D., Fok, K. F., Zutter, M. M., Adams, S. P., Rodriguez, B. A. & Santoro, S. A. (1991). Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen. Journal of Biological Chemistry 266, 7363-7367.[Abstract/Free Full Text]

Superti, F. & Donelli, G. (1995). Characterization of SA-11 rotavirus receptorial structures on human colon carcinoma cell line HT-29. Journal of Medical Virology 47, 421-428.[Medline]

Uciechowski, P. & Schmidt, R. E. (1989). Cluster report: CD11. In Leucocyte Typing IV, pp. 543-551. Edited by W. Knapp, B. Dorken, W. R. Gilks, E. P. Rieber, R. E. Schmidt, H. Stein & A. E. G. K. von dem Borne. Oxford: Oxford University Press.

Wasserman, K., Subklewe, M., Pothoff, G., Banik, N. & Schell-Frederick, E. (1994). Expression of surface markers on alveolar macrophages from symptomatic patients with HIV infection as detected by flow cytometry. Chest 105, 1324-1334.[Abstract]

Yeager, M., Berriman, J. A., Baker, T. S. & Bellamy, A. R. (1994). Three-dimensional structure of the rotavirus haemagglutinin VP4 by cryo-electron microscopy and difference map analysis. EMBO Journal 13, 1011-1018.[Abstract]

Received 9 March 2000; accepted 7 June 2000.