VLA-2 (alpha2beta1) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment

Abstract

In an attempt to identify the rotavirus receptor, we tested 46 cell lines of different species and tissue origins for susceptibility to infection by three N-acetyl-neuraminic (sialic) acid (SA)-dependent and five SA-independent rotavirus strains. Susceptibility to SA-dependent or SA-independent rotavirus infection varied depending on the cell line tested and the multiplicity of infection (MOI) used. Cells of renal or intestinal origin and transformed cell lines derived from breast, stomach, bone, or lung were all susceptible to rotavirus infection, indicating a wider host tissue range than previously appreciated. Chinese hamster ovary (CHO), baby hamster kidney (BHK-21), guinea pig colon (GPC-16), rat small intestine (Rie1), and mouse duodenum (MODE-K) cells were found to support only limited rotavirus replication even at MOIs of 100 or 500, but delivery of rotavirus particles into the cytoplasm by lipofection resulted in efficient rotavirus replication. The rotavirus cell attachment protein, the outer capsid spike protein VP4, contains the sequence GDE(A) recognized by the VLA-2 (alpha2beta1) integrin, and to test if VLA-2 is involved in rotavirus attachment and entry, we measured infection in CHO cells that lack VLA-2 and CHO cells transfected with the human alpha2 subunit (CHOalpha2) or with both the human alpha2 and beta1 subunits (CHOalpha2beta1) of VLA-2. Infection by SA-dependent or SA-independent rotavirus strains was 2- to 10-fold more productive in VLA-2-expressing CHO cells than in parental CHO cells, and the increased susceptibility to infection was blocked with anti-VLA-2 antibody. However, the levels of binding of rotavirus to CHO, CHOalpha2, and CHOalpha2beta1 cells were equivalent and were not increased over binding to susceptible monkey kidney (MA104) cells or human colonic adenocarcinoma (Caco-2, HT-29, and T-84) cells, and binding was not blocked by antibody to the human alpha2 subunit. Although the VLA-2 integrin promotes rotavirus infection in CHO cells, it is clear that the VLA-2 integrin alone is not responsible for rotavirus cell attachment and entry. Therefore, VLA-2 is not involved in the initial attachment of rotavirus to cells but may play a role at a postattachment level.

FIG. 1.

Detection of rotavirus antigen (fluorescent foci) in rhesus, African, or buffalo green monkey kidney cells (A); rhesus, rabbit, canine, bovine, or human kidney cells (B); human intestinal cells (C); human, porcine, or murine intestinal cells (D); human breast, bone, lung, cervix, or hepatic cells (E); and human rhabdomyosarcoma, murine, human, or rat fibroblasts, or murine metastatic prostate cancer cells (F). The infectivity of the SA-dependent RRV strain (solid) or the SA-independent WC3 strain (shaded) is expressed in FFU as measured by FFA. An MOI of 10 was used to infect all cells. As a control, cells incubated with medium alone were used (data not shown). The values shown are the arithmetic means of at least three replicate experiments. The error bars represent one standard error of the mean.

FIG. 2.

Detection of rotavirus antigen (fluorescent foci) in rhesus monkey kidney (MA104), Chinese hamster ovary (CHO-K-1, CHO-CD36, and CHO-p901), baby hamster kidney (BHK-21), and guinea pig (GPC-16), rat (Rie1), and murine (MODE-K) intestinal cells. The infectivity of the SA-dependent RRV strain or the SA-independent WC3 strain is expressed in FFU as measured by FFA. An MOI of 500 (RRV) or 100 (WC3) was used to infect all cells. As a control, cells incubated with medium alone were used (data not shown). The values shown are the arithmetic means of at least three replicate experiments. The error bars represent one standard error of the mean.

FIG. 3.

Liposome-mediated infection of cells by SA-dependent RRV strain (A) or SA-independent WC3 strain (B) with Lipofectamine plus reagent. Synthesis of RRV and WC3 rotavirus proteins was determined with 1 μg of CsCl-purified and EDTA-treated rotavirus DLPs/ml. No virus antigen was detected in cells inoculated with RRV or WC3 DLPs without Lipofectamine plus reagent or with Lipofectamine plus reagent alone (data not shown). The values shown are the arithmetic means of at least three replicate experiments. The error bars represent one standard error of the mean.

FIG. 4.

Inhibition of RRV and WC3 rotavirus infectivity following treatment of MA104 cells with a range of concentrations (0 to 20 μg/ml) of MAb AK-7, directed to the α2 subunit of VLA-2 (A), or inhibition of RRV or WC3 rotavirus infectivity in MA104 cells by MAbs (10 μg/ml) to integrin subunits α2 (AK-7), α4 (9F10), β1 (MAR4), β3 (VI-PL2), αvβ3 integrin (23C6), and α2 (AK-7) plus αvβ3 integrin (23C6), or Norwalk virus capsid (NV3901) (B). MOI = 10. Virus infectivity is expressed as the percentage of FFU in antibody-treated cells versus those obtained in control (199 medium-treated) cells. The values shown are the arithmetic means of at least three replicate experiments. The error bars represent one standard error of the mean. A significant (P < 0.05; Mann-Whitney U test) decrease in rotavirus infectivity following treatment of MA104 cells with each antibody is indicated by an asterisk.

FIG. 5.

Inhibition of RRV or WC3 rotavirus infectivity in MA104 cells treated with 20 mU of neuraminidase (NA) (A. ureafaciens)/ml and/or MAb (10 μg/ml) to the α2 integrin subunit (AK-7). MOI = 10. Virus infectivity is expressed as the percentage of FFU in neuraminidase-treated and/or antibody-treated cells versus those obtained in control (TNC buffer- and 199 medium-treated) cells. The values shown are the arithmetic means of at least three replicate experiments. The error bars represent one standard error of the mean. A significant (P < 0.05; Mann-Whitney U test) decrease in rotavirus infectivity following treatment of MA104 cells with neuraminidase (NA) and/or AK-7 MAb is indicated by an asterisk.

FIG. 6.

Flow cytometry histograms of the cell surface expression of integrin subunits α2 and β1 of VLA-2. The cell line analyzed is indicated in each histogram. Expression of α2 or β1 was determined by using 10 μg of MAb AK-7 (MA104, Caco-2, and CHO-p901) or MAb Ha1/29 (Rie1) or of MAb MAR4 (MA104, Caco-2, and CHO-p901) or MAb Ha2/5 (Rie1)/ml, respectively, in a single- or two-step stain. MAb NV3901, directed against the recombinant Norwalk virus capsid (35, 64), was used to match the isotype (IgG1) in a two-step stain.

FIG. 7.

(A) Flow cytometry histograms of the surface expression of human integrin subunits α2 and β1 of VLA-2 in CHOα2 and CHOα2β1 cells. The cell line analyzed is indicated in each histogram. Expression of α2 or β1 was determined by using 10 μg of MAb AK-7 or MAb MAR4/ml, respectively, in a single-step stain. MAb NV3901, directed against the recombinant Norwalk virus capsid (35, 64), was used to match the isotype (IgG1) in a two-step stain. (B) VLA-2 integrin promotes CHO cell infection. CHO-p901 or stably transfectant CHOα2 and CHOα2β1 cells were infected with SA-dependent RRV or SA-independent WC3 rotaviruses (MOI = 500 or 100, respectively). Virus infectivity is expressed as the percentage of FFU in CHOα2 and CHOα2β1 cells versus those obtained in CHO-p901 cells. The values shown are the arithmetic means of at least five replicate experiments. The error bars represent one standard error of the mean. A significant (P < 0.05; Mann-Whitney U test) increase in rotavirus infectivity of CHOα2 and CHOα2β1 cells with respect to infectivity of CHO-p901 cells is indicated by an asterisk.

FIG. 7.

(A) Flow cytometry histograms of the surface expression of human integrin subunits α2 and β1 of VLA-2 in CHOα2 and CHOα2β1 cells. The cell line analyzed is indicated in each histogram. Expression of α2 or β1 was determined by using 10 μg of MAb AK-7 or MAb MAR4/ml, respectively, in a single-step stain. MAb NV3901, directed against the recombinant Norwalk virus capsid (35, 64), was used to match the isotype (IgG1) in a two-step stain. (B) VLA-2 integrin promotes CHO cell infection. CHO-p901 or stably transfectant CHOα2 and CHOα2β1 cells were infected with SA-dependent RRV or SA-independent WC3 rotaviruses (MOI = 500 or 100, respectively). Virus infectivity is expressed as the percentage of FFU in CHOα2 and CHOα2β1 cells versus those obtained in CHO-p901 cells. The values shown are the arithmetic means of at least five replicate experiments. The error bars represent one standard error of the mean. A significant (P < 0.05; Mann-Whitney U test) increase in rotavirus infectivity of CHOα2 and CHOα2β1 cells with respect to infectivity of CHO-p901 cells is indicated by an asterisk.

FIG. 8.

Inhibition of RRV or WC3 rotavirus infectivity in stably transfectant CHOα2 and CHOα2β1 cells by MAb (10 μg/ml) to integrin subunit α2 (AK-7). MOI = 500 (RRV) or 100 (WC3). The data are expressed as the percentage of the virus infectivity obtained when the CHO-p901 cells were preincubated with α-MEM as a control. The values shown are the arithmetic means of at least three replicate experiments. The error bars represent one standard error of the mean. An asterisk indicates a significant (P < 0.05; Mann-Whitney U test) decrease in rotavirus infectivity of CHO-p901, CHOα2, or CHOα2β1 cells following treatment of the cells with antibody to the α2 or β1 subunit of VLA-2 integrin with respect to the infectivity obtained in corresponding untreated CHO-p901, CHOα2, or CHOα2β1 cells, respectively.

FIG. 9.

Capacities of different concentrations (0 to 40 μg/ml) of MAb AK-7, directed to the α2 subunit of the VLA-2 integrin, to inhibit binding of RRV or WC3 rotavirus strains in MA104 cells (A) and binding of infectious SA-dependent rotavirus (RRV) in the absence or presence of MAb AK-7 to intestinal (Caco-2, HT-29, and T-84), kidney (MA104), or ovary (CHO-K1, CHO-p901, CHOα2, and CHOα2β1) cells (B) or SA-independent rotavirus (WC3) in the absence or presence of MAb AK-7 to intestinal (Caco-2, HT-29, and T-84), kidney (MA104), or ovary (CHO-K1, CHO-p901, CHOα2, and CHOα2β1) cells (C). Binding of infectious rotavirus was blocked (90 to 100%) when the virus was preincubated with rabbit hyperimmune serum containing neutralizing antibodies to RRV or WC3 (data not shown). The data are expressed as the percentage of infectious virus binding when MA104 cells were preincubated with 199 medium as a control. The values shown are the arithmetic means of at least three replicate experiments. The error bars represent one standard error of the mean.